Modified immunoglobulin FC fusion protein and its use

Abstract

The present invention relates to novel immunoglobulin Fc domain variant proteins and uses thereof. When expressed in the form of fusion proteins with different biologically active proteins, the immunoglobulin Fc domain variant proteins according to the embodiments of the present invention not only prolong the in vivo half-life of the biologically active proteins, but also effectively inhibit effector functions (such as ADCC or CDC), thereby allowing to minimize unexpected side effects.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under the Paris Convention for the Protection of Industrial Property Rights to U.S. Patent Application No. 62 / 847,470, filed on May 14, 2019, the contents of which are incorporated by reference in their entirety.

[0003] background

[0004] The present invention relates to novel modified immunoglobulin Fc fusion proteins and uses thereof, and more particularly, to novel modified immunoglobulin Fc fusion proteins having increased half-life and capable of minimizing side effects (such as ADCC and CDC, which may be caused by antibody Fc fragments); and uses thereof.

[0005] Immunoglobulin (antibody) Fc domain is widely used as a carrier protein for various treatments and diagnostic proteins.Antibodies include two functionally independent parts, i.e., a variable domain that is bound to an antigen and is referred to as "Fab", and a constant domain that is related to effector functions (such as complement activation and attack by phagocytes) and is referred to as "Fc".The serum half-life of Fc is long, while the life span of Fab is short (Capon et al., Nature 337:525-531,1989).When combined with therapeutic proteins or peptides, the Fc domain can provide a longer half-life or integrate functions such as the combination of Fc receptors, the combination of protein A, the fixation of complement and the transfer of placenta.In particular, the longer half-life of this Fc domain is due to the binding affinity of neonatal Fc receptors (hereinafter referred to as "FcRn") .

[0006] So far, many technologies for fusing Fc domains and biologically active proteins have been developed to increase the half-life of biologically active proteins. As a representative example, etanercept (trade name: It is a fusion protein in which TNFα receptor 2 is linked to the Fc domain of IgG1 antibody (US Pat. No. 5,447,851). In addition to this example, there are some cytokines and growth hormones fused to the Fc domain of IgG antibody.

[0007] However, unlike cell surface receptors fused to the extracellular domain, the fusion of water-soluble proteins to IgG results in reduced biological activity compared to non-fused cytokines or growth factors. The chimeric protein exists as a dimer and causes steric hindrance when binding to a target molecule such as a receptor due to the presence of two active proteins in close proximity to each other. Therefore, there is a need to overcome this problem to produce an effective Fc fusion protein.

[0008] Another limitation of Fc fusion technology is unexpected immune response. The Fc domain of immunoglobulin also has effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In particular, 4 human IgG isotypes bind with different affinities to activating Fcγ receptors (FcγR, including FcγRI, FcγRIIa and FcγRIIIa), inhibitory FcγRIIb receptors and the first factor of complement (C1q), resulting in very different effector functions (Bruhns, P. et al., Blood 113 (16): 3716-3725, 2009). The binding of IgG to FcγR or C1q depends on the residues located in the hinge region and CH2 domain. However, when the therapeutic protein fused to the Fc domain is given to a subject in vivo, the effector function of the Fc domain will cause apoptosis, cytokine secretion or inflammation in the subject, and therefore it is necessary to suppress the effector function to reduce unwanted reactions, unless absolutely necessary.

[0009] To overcome the above-identified problems, some modified Fc domain proteins have been developed, including those described in US2006 / 0074225A1, Armour, KL et al. (Eur. J. Immunol., 29(8):2613-2624, 1999), Shields, RL et al. (J. Biol. Chem. 276(9):6591-6604, 2001), and Idusogie, EE. et al. (J. Immunol. 164(8):4178-4184, 2000).

[0010] Additionally, despite the improvements in half-life, there is still a need to further improve the half-life of therapeutic proteins fused to an Fc domain.

[0011] Overview

[0012] However, the above-mentioned prior arts have disadvantages of failing to achieve a satisfactory half-life extension level or having other side effects.

[0013] Therefore, the present invention is to solve the above-mentioned various problems, and the purpose of the present invention is to provide a novel modified Fc domain protein, which does not affect the production and activity of biologically active proteins, while merging without effector functions (such as ADCC and CDC) of therapeutic proteins to show a longer half-life. However, this purpose is exemplary and does not limit the scope of the present invention.

[0014] According to one aspect of the present invention, there is provided an immunoglobulin Fc domain variant protein, wherein the 18th and 196th amino acids of a modified IgG4 Fc domain protein comprising an amino acid sequence selected from SEQ ID NOs: 1-4 are mutated to other amino acids.

[0015] According to another aspect of the present invention, there is provided a fusion protein in which at least one biologically active protein (API, active pharmaceutical ingredient) is linked to the N-terminus and / or C-terminus of an immunoglobulin Fc domain variant protein.

[0016] According to still another aspect of the present invention, a polynucleotide encoding an immunoglobulin Fc domain variant protein or a fusion protein is provided.

[0017] According to still another aspect of the present invention, a homo- or hetero-dimer containing the fusion protein is provided.

[0018] According to still another aspect of the present invention, there is provided a composition containing a fusion protein, a polynucleotide encoding the fusion protein, or a homo- or hetero-dimer thereof as an active ingredient.

[0019] According to still another aspect of the present invention, a method for prolonging the in vivo half-life of a biologically active protein is provided, the method comprising the step of administering to a subject a fusion protein obtained by fusing the biologically active protein to an immunoglobulin Fc domain variant protein, or a homo- or hetero-dimer containing the fusion protein.

[0020] By being fused to another biologically active protein, the immunoglobulin Fc domain variant protein according to an exemplary embodiment of the present invention can increase the half-life of the biologically active protein and inactivate the antibody Fc domain-dependent effector function (such as the side effects ADCC and CDC caused when using antibody-based protein therapeutics). However, the effects of the present invention are not limited thereto. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1-3 A schematic diagram showing the structure of a monospecific fusion protein according to an exemplary embodiment of the present invention;

[0023] FIG4 is a schematic diagram showing the structure of a bispecific fusion protein according to an exemplary embodiment of the present invention;

[0024] Figure 5 is a schematic diagram showing the structure of a homodimer composed of a bispecific fusion protein according to an exemplary embodiment of the present invention;

[0025] Figure 6 A schematic diagram showing the structure of a homodimer of a monospecific fusion protein according to an exemplary embodiment of the present invention;

[0026] Figure 7 A schematic diagram showing the structure of a heterodimer of two different monospecific fusion proteins according to an exemplary embodiment of the present invention;

[0027] Figure 8 is a schematic diagram showing the structure of a heterodimer composed of two different bispecific fusion proteins according to an exemplary embodiment of the present invention;

[0028] Fig. 9 A schematic diagram showing the structure of a homodimer of a monospecific fusion protein having an IgG1 hinge region and a linker peptide according to an exemplary embodiment of the present invention;

[0029] Fig.10 is a schematic diagram showing the structure of a heterodimer composed of two different monospecific fusion proteins according to an exemplary embodiment of the present invention;

[0030] Fig.11 A schematic diagram showing the structure of a homodimer of a monospecific fusion protein having a biologically active protein at the C-terminus according to an exemplary embodiment of the present invention;

[0031] Fig.12 A schematic diagram showing the structure of a heterodimer composed of two different bispecific fusion proteins having a biologically active protein at the C-terminus according to an exemplary embodiment of the present invention;

[0032] Fig.13 is a schematic diagram schematically showing the process for recycling antibodies through FcRn;

[0033] Fig.14a is a photograph showing SDS-PAGE analysis results showing sample production results of a fusion protein (PG-11) according to an exemplary embodiment of the present invention, and Fig.14b is a chromatogram showing the results of PG-11 produced by size exclusion high performance liquid chromatography (SEC-HPLC);

[0034] Fig.15a is a photograph showing SDS-PAGE analysis results showing sample production results of a fusion protein (PG-22) according to an exemplary embodiment of the present invention, and Fig.15b is a chromatogram showing the results of PG-22 produced by size exclusion high performance liquid chromatography (SEC-HPLC);

[0035] Fig.16a is a photograph showing SDS-PAGE analysis results showing sample production results of a fusion protein (PG-088) according to an exemplary embodiment of the present invention, and Fig.16b is a chromatogram showing the results of PG-088 produced by size exclusion high performance liquid chromatography (SEC-HPLC);

[0036] Fig.17a is a photograph showing SDS-PAGE analysis results showing sample production results of a fusion protein (PG-088m) according to an exemplary embodiment of the present invention, and Fig.17b is a chromatogram showing the results of PG-088m produced by size exclusion high performance liquid chromatography (SEC-HPLC);

[0037] Fig.18a is a photograph showing SDS-PAGE analysis results showing sample production results of a fusion protein (PG-075) according to an exemplary embodiment of the present invention, and Fig.18b A photograph showing the results of western blot analysis of the produced PG-075 using an anti-IgG antibody;

[0038] Fig.19a is a photograph showing SDS-PAGE analysis results showing sample production results of a fusion protein (PG-110) according to an exemplary embodiment of the present invention, and Fig.19b is a chromatogram showing the results of PG-110 produced by size exclusion high performance liquid chromatography (SEC-HPLC);

[0039] Fig.20a is a photograph showing SDS-PAGE analysis results showing sample production results of a fusion protein (PG-129) according to an exemplary embodiment of the present invention, and Fig.20b is a chromatogram showing the results of PG-129 produced by size exclusion high performance liquid chromatography (SEC-HPLC);

[0040] Fig.21a is a photograph showing SDS-PAGE analysis results showing sample production results of a fusion protein (PG-400) according to an exemplary embodiment of the present invention, and Figure 21b is a chromatogram showing the results of PG-400 produced by size exclusion high performance liquid chromatography (SEC-HPLC);

[0041] Fig.22a is a photograph showing SDS-PAGE analysis results showing sample production results of a fusion protein (PG-410) according to an exemplary embodiment of the present invention, and Figure 22b is a chromatogram showing the results of PG-410 produced by size exclusion high performance liquid chromatography (SEC-HPLC);

[0042] Fig.23 A photograph showing SDS-PAGE analysis results showing sample production results of heterodimeric proteins MG12-4 (left side) and MG12-5 (right side) according to an exemplary embodiment of the present invention;

[0043] Fig.24a To show photographs showing SDS-PAGE analysis results showing sample production results of a fusion protein (PG-50-1) according to an exemplary embodiment of the present invention, Figure 24b To show photographs showing SDS-PAGE analysis results showing sample production results of a fusion protein (PG-50-2) according to an exemplary embodiment of the present invention, Fig.24c A photograph showing SDS-PAGE analysis results showing sample production results of a fusion protein (PG-073) according to an exemplary embodiment of the present invention (left side) and a schematic diagram showing structures of various cleavage forms of the fusion protein estimated based on the SDS-PAGE analysis results (right side) are shown, Fig.24d A photograph showing the results of SDS-PAGE analysis of a sample of a fusion protein (PG-210-5) according to an exemplary embodiment of the present invention (upper portion) and a chromatogram showing the results of analyzing the generated PG-210-5 by size exclusion high performance liquid chromatography (SEC-HPLC) (lower portion), and Fig.24e A photograph showing the results of SDS-PAGE analysis showing the sample production results of the fusion protein (PG-210-6) according to an exemplary embodiment of the present invention (upper part) and a chromatogram showing the results of analyzing the produced PG-210-6 by size exclusion high performance liquid chromatography (SEC-HPLC) (lower part);

[0044] Fig.25 is a series of graphs showing the results of analyzing the binding affinity of the fusion protein (PG-11) prepared according to an exemplary embodiment of the present invention to the recombinant human neonatal Fc receptor (rhFcRn) by using surface plasmon resonance (SPR) analysis, and each of the three graphs shows the results of independent testing;

[0045] Fig.26 A series of graphs showing the results of analyzing the binding affinity of a fusion protein (PG-11, upper part) prepared according to an exemplary embodiment of the present invention to a recombinant human neonatal Fc receptor (rhFcRn) by using surface plasmon resonance (SPR) analysis, compared with the case of Trulicity (lower part) using an IgG4 Fc domain, and the left, middle and right graphs respectively show the results of 3 independent tests;

[0046] Fig. 27 A series of graphs showing the results of analyzing the binding affinity of a fusion protein (PG-11, upper part) prepared according to an exemplary embodiment of the present invention to a recombinant human neonatal Fc receptor (rhFcRn) by using surface plasmon resonance (SPR) analysis, in comparison with the case of a recombinant humanized antibody rituximab (Rituximab, lower part) containing an IgG1 Fc domain, and the graphs on the left, middle and right sides of the lower part respectively show the results of 3 independent tests;

[0047] Fig.28 is a series of graphs showing comparative analysis results of the binding affinity of rhFcRn to each of a bispecific fusion protein (in which an anti-PD-L1 scFv and an IL-2 protein are linked to the N-terminus and C-terminus of a NTIG protein according to an exemplary embodiment of the present invention, respectively) (left side) and a bispecific fusion protein (in which an anti-PD-L1 scFv and an IL-2 protein are linked to the N-terminus and C-terminus of a modified IgG4 Fc protein of SEQ ID NO: 2, respectively) (right side) as a control group through SPR analysis using rhFcRn as a ligand and two substances as samples;

[0048] Fig.29 A series of graphs showing the Fig.28 SPR analysis using two substances as ligands and rhFcRn as a sample in the opposite manner, comparative analysis results of the binding affinity of rhFcRn to each of a bispecific fusion protein (in which an anti-PD-L1 scFv and an IL-2 protein are linked to the N-terminus and C-terminus of an NTIG protein according to an exemplary embodiment of the present invention, respectively) (left side) and a bispecific fusion protein (in which an anti-PD-L1 scFv and an IL-2 protein are linked to the N-terminus and C-terminus of a modified IgG4 Fc protein of SEQ ID NO: 2, respectively) (right side) as a control group;

[0049] Fig.30 A series of graphs showing the results of analyzing the binding affinity of the fusion protein (PG-11) according to an exemplary embodiment of the present invention to Fcγ receptor I (FcγR1, left side) or Fcγ receptor IIIa (FcγR3a, right side) by BLI analysis, compared with the case of IgG1-based humanized antibody rituximab, and the graphs in the upper, middle and lower parts respectively show the results of 3 independent tests;

[0050] Fig.31A series of graphs showing the results of analyzing the binding affinity of the fusion protein (PG-11) according to an exemplary embodiment of the present invention to various other Fcγ receptors (FcγRIIa, FcγRIIb and FcγRIIIb) by BLI analysis, compared with the case of IgG1-based humanized antibody rituximab, and using PBS as a control group;

[0051] Fig.32 A graph showing the results of analyzing the binding affinity of the fusion protein (PG-11) according to an exemplary embodiment of the present invention to complement C1q in comparison with that of rituximab;

[0052] Fig.33 is a series of graphs showing the results of comparative analysis of GLP-2 activity of fusion proteins (PG-22, PG-201-5, PG-201-6, MG12-4 and MG12-5) containing GLP-2 analogs according to various exemplary embodiments of the present invention, and the two graphs in the upper and lower parts respectively show the results of two independent tests;

[0053] Fig.34 is a series of graphs showing the results of examining the affinity of the treated concentrations of the dimeric IL-10 variant fusion protein according to Example 2-3 (left side) and the monomeric IL-10 variant fusion protein according to Example 2-4 (right side) to IL-10R1 by BLI analysis, wherein the fusion protein is according to the present invention;

[0054] Fig.35a This is a graph showing the results of quantitatively analyzing the release level of TNF-α in macrophages by ELISA analysis according to the concentration of the fusion protein PG-210-6 according to an exemplary embodiment of the present invention. Fig.35b A graph showing the results of quantitatively analyzing the release level of TNF-α in macrophages by ELISA analysis according to the treatment concentration of the fusion protein PG-410 according to an exemplary embodiment of the present invention, and Fig.35c A graph showing the results of quantitatively analyzing the release level of TNF-α in macrophages by ELISA analysis according to the treatment concentrations of the fusion proteins PG-110 and PG-088m according to an exemplary embodiment of the present invention;

[0055] Fig.36a A series of graphs showing the results of analyzing the affinity of FcεRIα-modified IgG4 Fc (upper part) as a comparative example and the fusion protein FcεRIα-NTIG-IL-10Vm (lower part) of Example 3-3 of the present invention to mouse IgE by BLI analysis, and Fig.36bThis is a graph showing the results of analyzing the affinity of the fusion protein FcεRIα-NTIG-IL-10Vm of Example 3-3 of the present invention to human IgE by BLI analysis;

[0056] Fig.37 A graph showing the results of analyzing the IgE inhibitory activity of the fusion protein PG-110 according to an exemplary embodiment of the present invention and the FcεRIα-modified IgG4 Fc according to an exemplary embodiment of the present invention by β-hexosaminidase release assay;

[0057] Fig.38 A graph showing the results of analyzing the binding affinity of the fusion proteins PG-400 (left side) and PG-410 (right side) according to an exemplary embodiment of the present invention to CD40L by BLI analysis; and

[0058] Fig.39 It is a graph showing the results of analyzing the pharmacokinetic characteristics of various administration routes (SC, IV, IP and IM) of the fusion protein PG-110 according to an exemplary embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0060] According to one aspect of the present invention, there is provided an immunoglobulin Fc domain variant protein, wherein the 18th and 196th amino acids of a modified IgG4 Fc domain protein comprising an amino acid sequence selected from SEQ ID NOs: 1-4 are mutated to other amino acids.

[0061] The term "modified IgG4 immunoglobulin Fc domain protein" or "modified IgG4 Fc domain protein" as used herein refers to a recombinant antibody Fc domain protein prepared by combining all or part of hinge, CH2 and CH3, which are Fc domain construction elements of immunoglobulins, with those elements derived from different types of antibody molecules (i.e., IgG, IgD, IgE and IgM). As representative modified IgG4 immunoglobulin Fc domain proteins, those disclosed in Korean Patent No. 897938 are mentioned.

[0062] As used herein, the term "immunoglobulin Fc domain variant protein" or "Fc domain variant protein" refers to a mutant protein in which at least one amino acid in a modified IgG4 Fc domain protein is substituted, deleted or added.

[0063] In the immunoglobulin Fc domain variant protein, the mutation of the 18th amino acid may be a mutation selected from T18N, T18K and T18Q, the mutation of the 196th amino acid may be a mutation selected from M196A, M196F, M196I, M196L, M196V, M196P and M196W, the mutation of the 18th amino acid is more preferably T18Q, and the mutation of the 196th amino acid is preferably M196L.

[0064] The lysine (K) at the C-terminus of the immunoglobulin Fc domain variant protein may or may not be removed. Whether to remove the lysine can be determined based on whether another API is connected to the C-terminus. For example, when the API is connected to the C-terminus of the immunoglobulin Fc domain variant protein of the present invention, lysine removal may be advantageous in terms of protein production, while when the API is not connected to the C-terminus, lysine removal is not required.

[0065] The immunoglobulin Fc domain variant protein may contain an amino acid sequence selected from SEQ ID NOs: 5-9.

[0066] According to another aspect of the present invention, there is provided a fusion protein (API) in which at least one biologically active protein (API, active pharmaceutical ingredient) is linked to the N-terminus and / or C-terminus of an immunoglobulin Fc domain variant protein. Figure 1 -4).

[0067] The term "biologically active protein" as used herein is a concept that is distinguished from structural proteins that are proteins that constitute human tissues and organs, and refers to proteins that perform specific functions (such as metabolism and intercellular or intracellular signaling that occurs in the human body), and can be used to diagnose and / or treat diseases caused by a lack or excess of the protein.

[0068] like Figure 1 As shown in Figure 4, in the fusion protein (10-40) of the present invention, the biologically active protein or active pharmaceutical ingredient (API, 11) can be connected to the N-terminus or C-terminus of the immunoglobulin Fc domain variant protein (12) through the hinge region (13) of IgG1 and / or a linker peptide (14) such as a G4S linker. However, the hinge region (13) is essential for dimerization. When the biologically active protein (11) is directly connected to the hinge region (13) of IgG1, the hinge region (i.e., SEQ ID NOs: 20 and 21) can be regarded as Figure 3 The hinge region provides flexibility and a dimerization moiety capable of forming intermolecular disulfide bonds via cysteine ​​residues.

[0069] The fusion protein according to an exemplary embodiment of the present invention may contain one or two biologically active proteins. When the fusion protein contains two biologically active proteins, the fusion protein itself exhibits bispecificity ( FIG. 4 ). Figure 4a As shown, the first biologically active protein (11a) is connected to the N-terminus of the immunoglobulin Fc domain variant protein (12) through the hinge region (13) of IgG1, and the second biologically active protein (11b) is connected to the C-terminus of the immunoglobulin Fc domain variant protein (12) through a linker peptide (14). In addition, as Figure 4b As shown, a linker peptide (14) such as a G4S linker is additionally connected to the N-terminus of the hinge region (13) of IgG1, a first biologically active protein (11a) is connected to the N-terminus of the linker peptide (14), and a second biologically active protein (11b) is connected to the C-terminus of the immunoglobulin Fc domain variant protein (12) via the linker peptide (14). In this case, the linker peptide (14) connected to the N-terminus and the linker peptide (14) connected to the C-terminus may have the same sequence, or the linker peptide has the same properties as the G4S linker but may be partially different in specific sequence.

[0070] In a fusion protein, the biologically active protein can be a cytokine, an enzyme, a coagulation factor, an extracellular domain of a membrane receptor, a growth factor, a peptide hormone, or an antibody mimetic.

[0071] In the fusion protein, the length of the linker peptide may be 2-60a.a., 4-55a.a., 5-50a.a., 5-46a.a., 5-45a.a. or 5-30a.a. In the linker peptide, the linker peptide that connects the API to the N-terminus of the immunoglobulin Fc domain variant protein may include a part or all of the IgG1 antibody heavy chain hinge region, and the case where the linker peptide includes a part of the hinge region may be a case where an artificial linker peptide is added to the N-terminus or C-terminus of a part of the hinge region. In addition, the hinge region may be a hybrid hinge region in which parts of the hinge regions of two or more heavy chains of an antibody are mixed. Specifically, the linker peptide can be a combination of one or more selected from the following sequences: GGGGSGGGGSGGGGSEKEKEEQEERTHTCPPCP (SEQ ID NO: 14), RNTGRGGEEKKGSKEKEEQEERETKTPECP (SEQ ID NO: 15), GGGGSGGGGSGGGGSEPKSCDKTHTCPPCP (SEQ ID NO: 16), GSGGGSGTLVTVSSESKYGPPCPPCP (SEQ ID NO: 17), GGGGSGGGGSGGGGSEPKSSDKTHTCPPCP (SEQ ID NO: 18), EPKSSDKTHTCPPCP (SEQ ID NO: 19), EPKSCDKTHTCPPCP (SEQ ID NO: 20), GGGGSGGGGSGGGGSAKNTTAPATTRNTTRGGEEKKKEKEKEEQEERTHTCPPCP (SEQ ID NO: 21), IDNO:21), A(EAAAK)4ALEA(EAAAK)4A(SEQ ID NO:22), (G4S) n (Unit sequence: SEQ ID NO: 23, n is an integer from 1 to 10), (GSSGGS) n (Unit: SEQ ID NO: 24, n is an integer from 1 to 10), SGGGSGGGGSGGGGSGGEEQEEGGS (SEQ ID NO: 25), AAGSGGGGGSGGGGSGGGGS (SEQ ID NO: 26), KESGSSVSSEQLAQFRSLD (SEQ ID NO: 27), EGKSSGSGSESKST (SEQ ID NO: 28), GSAGSAAGSGEF (SEQ ID NO: 28) NO:29), (EAAAK) n(Specifies: SEQ ID NO:30, ranges 1-10), CRRRRRREAEAC(SEQ IDNO:31), GGGGGGGG(SEQ ID NO:32), GGGGGG(SEQ ID NO:33), PAPAP(SEQ ID NO:34), (Ala-Pro) n (embodies 1-10), VSQTSKLTRAETVFPDV(SEQ ID NO:35), PLGLWA(SEQ ID NO:36), TRHRQPRGWE(SEQ ID NO:37), AGNRVRRSVG(SEQ ID NO:38), RRRRRRRR(SEQ ID). NO:39)、GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE(SEQ ID NO:40)、GGGGSGGGGSGGGGS(SEQ ID NO:41)、AEAAAKEAAAAKA(SEQ ID NO:42)、GFLG(SEQ ID NO:41). NO:43) AKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECP(SEQ ID NO:44), GGSGG(SEQ ID NO:45), GGSGGSGGS(SEQ ID NO:46), GGGSGG(SEQ ID NO:47), and GSTSGSGKPGSGEGS(SEQ ID NO:48).

[0072] In this case, the cytokine may be a chemokine, an interferon, an interleukin, a colony stimulating factor or a tumor necrosis factor, the chemokine may be CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL16, XCL1, XCL2 or CX3CL1, and the interferon may be a stem cell factor. The interferon-α, interferon-β, interferon-ε, interferon-κ, interferon-δ, interferon-γ, interferon-τ, interferon-ω, interferon-ν or interferon-ζ, the interleukin may be IL-2, IL-4, IL-7, IL-10, IL-12α, IL-12β, IL-13, IL-15 or IL-21, the colony stimulating factor may be macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF) or promegapoietin, and the tumor necrosis factor may be TNFα, TNFβ, CD40 ligand (CD40L), Fas ligand (FasL), TNF-related apoptosis-inducing ligand (TRAIL) or LIGHT (tumor necrosis factor tumor necrosis factor superfamily member 14, TNFSF14).

[0073] In the fusion protein, the enzyme can be tissue-type plasminogen activator (tPA), urokinase, alteplase, reteplase, tenecteplase, streptokinase, anlsteplase, straphylokinase, nattokinase, limbrokinase, collagenase, glutenase, alglucerase, velaglucerase, imiglucerase, taliglucerase-α, β-glucocerebrosidase, α-galactosiase A, A), α-glucosidase, α-L-iduronidase, arylsulphatase B, agalsidase, adenosine deaminase, phenylalanine ammonia lyase, dornase, rasburicase, pegloticase, glucarpidase or ocriplasmin, but are not limited thereto.

[0074] In the fusion protein, the coagulation factor can be Factor VIII or Factor IX.

[0075] In the fusion protein, the membrane receptor can be a receptor tyrosine kinase (RTK) superfamily, a T cell receptor, an Fc receptor, a chemokine receptor, a Toll-like receptor family, a guanylate cyclase-coupled receptor (GCCR) or a receptor serine / threonine kinase, and the receptor tyrosine kinase can be an EGFR family, an insulin receptor family, a platelet-derived growth factor receptor (PDGFR) family, a vascular endothelial growth factor receptor (VEGFR) family, a fibroblast growth factor receptor (FGFR) family, a colon cancer kinase (CCK) family, a nerve growth factor receptor (NGFR) family, a hepatocyte growth factor receptor (HGFR) family, an erythropoietin-producing human hepatocyte receptor (EphR) family, a tyrosine protein kinase receptor (AXL) family, angiopoietin receptor (TIER) family, a receptor tyrosine kinase-related receptor (RYKR) family, a discoidin domain receptor (DDR) family, a rearranged in transfection (RET) family. family, leukocyte receptor tyrosine kinase (LTK) family, receptor tyrosine kinase-like orphan receptor (ROR) family, muscle-specific kinase (MuSK) family, CD80 (B7.1) or CD83, the Fc receptor can be an Fcγ receptor, an Fcα receptor or an Fcε receptor, the cytokine receptor can be a type 1 cytokine receptor, a type 2 cytokine receptor, a tumor necrosis factor receptor family, a chemokine receptor or a TGF-β receptor family, the Toll-like receptor family can be TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, the guanylate cyclase-coupled receptor can be natriuretic factor receptor 1 (NPR1), NPR2 or NPR3, and the receptor serine / threonine kinase can be a TGF-β superfamily receptor, a bone morphogenetic protein (BMP) receptor or an activin receptor-like kinase (ALK).

[0076] In the fusion protein, the growth factor can be adrenomedullin, angiogenin, bone morphogenetic protein, ciliary neurotrophic factor, leukemia inhibitory factor, epidermal growth factor (EGF), ephrin, erythropoietin (EPO), fibroblast growth factor (FGF), glial cell line-derived neurotrophic factor (GDNF) family, growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin, insulin-like growth factor (IGF), keratinocyte growth factor (KGF), migration stimulating factor (MSF), macrophage stimulating factor, neuregulin, neurotrophic factor, placental growth factor (PGF), platelet-derived growth factor (PDGF), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factor (TGF) family or vascular endothelial growth factor (VEGF).

[0077] In the fusion protein, the peptide hormone can be human growth hormone (hGH), a GLP-1 analog, a GLP-2 analog, insulin, adrenocorticotropic hormone, amylin, angiotensin, neuropeptide Y, enkephalin, neurotensin, atrial natriuretic peptide, calcitonin, cholecystokinin, gastrin, ghelin, glucagon, follicle-stimulating hormone, leptin, melanocyte-stimulating hormone (MSH), oxytocin, parathyroid hormone, prolactin, renin, somatostatin, thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), vasopressin, or vasoactive intestinal polypeptide.

[0078] In the fusion protein, the antibody mimetic can be scFv, V NAR 、V H H. affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, Fynomer, Kunitz domain peptide, monobody, repebody, VLR or nanoCLAMP.

[0079] In the fusion protein, the IL-10 protein may be an IL 10 variant protein in which the 87th amino acid isoleucine is substituted with alanine, or a monomeric IL-10 variant protein in which a linker peptide with a length of 6-10 a.a. is inserted between the 134th amino acid asparagine and the 135th amino acid lysine, and may include the amino acid sequence shown in SEQ ID NO: 54 or 56.

[0080] As used herein, the term "fusion protein" refers to a recombinant protein in which two or more proteins or domains responsible for a specific function in a protein are connected so that each protein or domain is responsible for its original function. A linker peptide with a flexible structure can usually be inserted between two or more proteins or domains. As linker peptides, GGGGSGGGGSGGGGSEKEKEEQEERTHTCPPCP (SEQ ID NO: 14), RNTGRGGEEKKGSKEKEEQEERETKTPECP (SEQ ID NO: 15), GGGGSGGGGSGGGGSEPKSCDKTHTCPPCP (SEQ ID NO: 16), GSGGGSGTLVTVSSESKYGPPCPPCP (SEQ ID NO: 15) NO: 17), GGGGSGGGGSGGGGSEPKSSDKTHTCPPCP (SEQ ID NO: 18), EPKSSDKTHTCPPCP (SEQ ID NO: 19), EPKSCDKTHTCPPCP (SEQ ID NO: 20), GGGGSGGGGSGGGGSAKNTTAPATTRNTTRGGEEEKKKEKEKEEQEERTHTCPPCP (SEQ IDNO:21), A(EAAAK)4ALEA(EAAAK)4A(SEQ ID NO:22)、(G4S) n (Unit sequence: SEQ ID NO: 23, n is an integer from 1 to 10), (GSSGGS) n (Unit: SEQ ID NO: 24, n is an integer from 1 to 10), SGGGSGGGGSGGGGSGGEEQEEGGS (SEQ ID NO: 25), AAGSGGGGGSGGGGSGGGGS (SEQ ID NO: 26), KESGSSVSSEQLAQFRSLD (SEQ ID NO: 27), EGKSSGSGSESKST (SEQ ID NO: 28), GSAGSAAGSGEF (SEQ ID NO: 28) NO:29), (EAAAK) n (unit: SEQ ID NO: 30, n is an integer of 1-10), CRRRRREAEAC (SEQ ID NO: 31), GGGGGGGG (SEQ ID NO: 32), GGGGGG (SEQ ID NO: 33), PAPAP (SEQ ID NO: 34), (Ala-Pro) n(n is an integer from 1 to 10), VSQTSKLTRAETVFPDV (SEQ ID NO:35), PLGLWA (SEQ ID NO:36), TRHRQPRGWE (SEQ ID NO:37), AGNRVRRSVG (SEQ ID NO:38), RRRRRRRR (SEQ ID NO:39), GSSGGGSSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO:40), GGGGSGGGGSGGGGS (SEQ ID NO:41), AEAAAKEAAAAKA (SEQ ID NO:42), GFLG (SEQ ID NO:43), AKATTAPATTRNTGRGGEKKKEKEKEEQEERETKTPECP (SEQ ID NO:44), GGSGG (SEQ ID NO:45), GGSGGSGGS (SEQ ID NO:46), GGGSGG (SEQ ID NO:47) and GSTGSSGKPGSGEGS (SEQ ID NO:48) may be mentioned.

[0081] The term "antibody" as used herein refers to an immunoglobulin molecule, which is a heterotetrameric protein produced by the combination of two identical heavy chains and two identical light chains, and is bound by the variable region (V L ) and the heavy chain variable region (V H ) to carry out antigen-specific binding, thereby causing antigen-specific humoral immune response. As antibodies, IgA, IgG, IgD, IgE, IgM, IgY, etc. can be mentioned according to their sources.

[0082] As used herein, the term "antigen-binding fragment of an antibody" refers to a fragment having antigen-binding ability derived from an antibody, and includes both fragments produced by cleaving an antibody with a protein cleavage enzyme and single-chain fragments produced in a recombinant manner, and examples thereof include Fab, F(ab')2, scFv, diabody, triabody, sdAb or V H H.

[0083] The term "Fab" as used herein refers to an antigen-binding antibody fragment (an antigen-binding fragment) produced by cleaving an antibody molecule with papain, which is a protease, and is composed of two peptide V H -CH1 and V L -C L The other fragment produced by papain is called Fc (fragment crystallizable).

[0084] The term "F(ab')2" as used herein refers to a fragment including an antigen binding site among fragments produced by cleaving an antibody with pepsin, which is a protease, and is in the form of a tetramer in which two Fab's are linked by a disulfide bond. Another fragment produced by pepsin is called pFc'.

[0085] The term "Fab" as used herein refers to a molecule having a similar structure to Fab produced by separating the above-mentioned F(ab')2 under weak reducing conditions.

[0086] The term "scFv" as used herein is an abbreviation for "single-chain variable fragment" and refers to a fragment which is not a fragment of an actual antibody but a fragment in which the heavy chain variable region (V H ) is connected to the light chain variable region (V L ) and is known to have antigen binding ability even though the fragment is not a specific antibody fragment (Glockshuber et al., Biochem. 29(6):1362-1367, 1990).

[0087] The terms "diabody" and "triabody" as used herein refer to antibody fragments in the form of two and three scFvs, respectively, connected by a linker.

[0088] The term "single domain antibody (sdAb)" as used herein refers to an antibody fragment also called a nanobody and composed of a single variable region fragment of an antibody. sdAbs derived from heavy chains are mainly used, but there are also reports that single variable region fragments derived from light chains specifically bind to antigens. Unlike conventional antibodies composed of heavy and light chains, V domain fragments composed of variable region fragments of shark antibodies are NAR and V composed of variable region fragments of camelid antibodies H H consists only of a single-chain dimer and is also included in sdAb.

[0089] The term "antibody mimetics" as used herein refers to a concept that includes proteins (such as monomeric antibodies and variable lymphocyte receptors (VLRs)) that have similar functions to antibodies (i.e., have antigen binding ability) prepared from protein scaffolds of non-antibody origin, which are different from normal full-length antibodies in which two heavy chains and two light chains form a quaternary structure of heterotetramers to exhibit functions.Examples of such antibody mimetics include affibodies derived from the Z domain of protein A (Nygren, PA, FEBS J. 275(11):2668-2676, 2008), affilins derived from γ-B crystallin or ubiquitin (Ebersbach et al., J. Mol. Biol. 372(1):172-185, 2007), affimers derived from cystatin (Johnson et al., Anal. Chem. 84(15):6553-6560, 2012), affitins derived from Sac7d (Krehenbrink et al., J. Mol. Biol. 383(5):1058-1068, 2008), alphabodies derived from triple helical coiled-coil proteins (Desmet et al., Nat. Commun. 5:5237, 2014), affimers derived from lipid transporters (Johnson et al., Anal. Chem. 84(15):6553-6560, 2012), affitins derived from Sac7d (Krehenbrink et al., J. Mol. Biol. 383(5):1058-1068, 2008), affimers derived from lipid transporters (Desmet et al., Nat. Commun. 5:5237, 2014), Anticarriers derived from apoptotic proteins (Skerra et al., FEBS J. 275(11):2677-2683, 2008), high-affinity multimers derived from various membrane receptor domains (Silverman et al., Nat. Biotechnol. 23(12):1556-1561, 2005), DARPins derived from ankyrin repeat motifs (Stumpp et al., Drug Discov. Today. 13(15-16):695-701, 2008), finoids derived from the SH3 domain of Fyn protein (Grabulovski et al., J. Biol. Chem. 282(5):3196-3204, 2007), Kunitz domain peptides derived from the Kunitz domains of various protein inhibitors (Nixon and Wood, Curr. Opin. Drug Discov. Dev. 9(2): 261-268, 2006), a monomeric antibody derived from the 10th domain of fibronectin type 3 (Koide and Koide, Methods Mol. Biol. 352: 95-109, 2007), a nanoCLAMP derived from the carbohydrate binding module 32-2 (Suderman et al., Protein Exp. Purif. 134: 114-124, 2017), a variable lymphocyte receptor (VLR) derived from hagfish (Boehm et al., Ann. Rev. Immunol. 30: 203-220, 2012), and a repebody based on VLR that has been modified to enhance antigen affinity (Lee et al., Proc. Natl. Acad. Sci. USA, 109: 3299-3304, 2012).

[0090] According to still another aspect of the present invention, a polynucleotide encoding an immunoglobulin Fc domain variant protein or a fusion protein is provided.

[0091] The polynucleotide may be any polynucleotide, as long as the polynucleotide is a polynucleotide encoding a protein containing an amino acid sequence selected from SEQ ID NO: 5-9, and may have codons optimized for the host cell type used to produce the protein. Such techniques for optimizing codons according to host cells are well known in the art (Fuglsang et al., Protein Expr. Purif. 31 (2): 247-249, 2003). The polynucleotide more preferably contains a nucleic acid sequence selected from SEQ ID NO: 10-13.

[0092] According to still another aspect of the present invention, a recombinant vector containing a polynucleotide is provided.

[0093] In the recombinant vector, the polynucleotide may be contained in the form of a gene construct operably linked to a regulatory sequence.

[0094] As used herein, the term "operably linked to" means that a target nucleic acid sequence (eg, in an in vitro transcription / translation system or a host cell) is linked to a regulatory sequence in such a way that the target nucleic acid sequence can be expressed.

[0095] As used herein, the term "regulatory sequence" is meant to include promoters, enhancers and other regulatory elements (e.g., polyadenylation signals). Examples of regulatory sequences include sequences that instruct the sustained expression of target nucleic acids in many host cells, sequences that instruct the expression of target nucleic acids only in specific tissue cells (e.g., tissue-specific regulatory sequences), and sequences that instruct the induction of expression by specific signals (e.g., inducible regulatory sequences). It will be appreciated by those skilled in the art that the design of expression vectors may vary according to factors such as the selection of host cells to be transformed and the desired protein expression level. The expression vector of the present invention may be introduced into host cells to express fusion proteins. Regulatory sequences that allow expression in eukaryotic cells and prokaryotic cells are well known to those skilled in the art. As described above, these regulatory sequences typically include regulatory sequences responsible for transcription initiation, and optionally, poly-A signals responsible for transcription termination and stabilization of transcripts. In addition to transcriptional regulatory factors, additional regulatory sequences may include translational enhancers and / or naturally combined or heterologous promoter regions. For example, possible regulatory sequences that allow expression in mammalian host cells include CMV-HSV thymidine kinase promoter, SV40, RSV promoter (Rous sarcoma virus), human kidney urea 1α-promoter, MMTV promoter (Moloney mouse tumor virus) that induces glucocorticoids, metallothionein or tetracycline inducible promoters or amplifiers (such as CMV amplifier and SV40 amplifier). It is believed that for expression in neural cells, neurofilament promoter, PGDF promoter, NSE promoter, PrP promoter or thy-1 promoter can be used. The above promoters are known in the art and are described in the literature (Charron, J. Biol. Chem. 270: 25739-25745, 1995). For expression in prokaryotic cells, many promoters have been disclosed, including lac promoter, tac promoter or trp promoter. In addition to factors capable of initiating transcription, the regulatory sequence may include a transcription termination signal such as SV40-poly-A site and TK-poly-A site located downstream of the polynucleotide according to an exemplary embodiment of the present invention.In this specification, suitable expression vectors are known in the art, and examples thereof include Okayama-Berg cDNA expression vector pcDV1 (Parmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pSPORT1 (GIBCO BRL), pGX-27 (Korean Patent No. 1442254), pX (Pagano et al., Science 255: 1144-1147, 1992), yeast two-hybrid vectors (such as pEG202 and dpJG4-5 (Gyuris et al., Cell 75: 791-803, 1995)) and prokaryotic expression vectors (such as λgt11 and pGEX (Amersham Pharmacia)). In addition to the nucleic acid molecule of the present invention, the vector may further include a polynucleotide encoding a secretion signal. The secretion signal is well known to those skilled in the art. In addition, depending on the expression system used, a leader sequence that directs the fusion protein according to an exemplary embodiment of the present invention to a cellular compartment can be combined with a polynucleotide coding sequence according to an exemplary embodiment of the present invention, and is preferably a leader sequence that is capable of secreting the decoded protein or its protein directly into the periplasm or the extracellular medium.

[0096] In addition, the carrier of the present invention can be prepared, for example, by standard recombinant DNA technology, and examples of standard recombinant DNA technology include connection of smooth ends and adhesion ends, restriction endonuclease treatment to provide appropriate ends, removal of phosphate groups by alkaline phosphatase treatment to prevent inappropriate binding and enzymatic connection by T4 DNA ligase. The carrier of the present invention can be prepared by recombining a signal peptide obtained by chemical synthesis or genetic recombination technology, a DNA encoding an immunoglobulin Fc domain variant protein according to an exemplary embodiment of the present invention, or a DNA encoding a fusion protein containing them with a carrier comprising an appropriate regulatory sequence. The carrier containing the regulatory sequence can be purchased or prepared commercially, and in an exemplary embodiment of the present invention, a pBispecific backbone carrier (Genexine, Inc., Korea), a pAD15 carrier, a pGP30 (Genexine, Inc. Korea) or a pN293F carrier (Y-Biologics, Inc., Korea) is used as a backbone carrier.

[0097] The expression vector may further include a polynucleotide encoding a secretion signal sequence, and the secretion signal sequence induces extracellular secretion of the recombinant protein expressed in the cell, and may be a tissue plasminogen activator (tPA) signal sequence, a herpes simplex virus glycoprotein Ds (HSV gDs) signal sequence, or a growth hormone signal sequence.

[0098] The expression vector according to an exemplary embodiment of the present invention may be an expression vector capable of expressing a protein in a host cell, and the expression vector may be in any form, such as a plasmid vector, a viral vector, a cosmid vector, a phagemid vector, or an artificial human chromosome.

[0099] According to still another aspect of the present invention, a homo- or hetero-dimer containing the fusion protein is provided.

[0100] In homodimers, such as Figure 5 As shown, when different biologically active proteins (11a and 11b) are linked to the N-terminus and C-terminus of the immunoglobulin Fc domain variant protein (12), respectively, the prepared homodimer (100) exhibits bispecificity. Figure 6 As shown, when only one monospecific fusion protein containing a biologically active protein (11) is dimerized, the resulting homodimer (200) still exhibits monospecificity ( Figure 6 ). In the case of heterodimers, e.g. Figure 7 As shown, when two different monospecific fusion proteins containing biologically active proteins (11a and 11b) are dimerized, the prepared heterodimer (300) exhibits bispecificity. Figure 8 As shown, when two different bispecific fusion proteins, each containing two different biologically active proteins (11a, 11b, 11c, and 11d), are dimerized, the resulting heterodimer (400) exhibits tetraspecificity.

[0101] Optionally, the homo- or hetero-dimers (500, 600, 700 and 800) according to an exemplary embodiment of the present invention may contain one or more linker peptides (14) between the biologically active protein (11, 11a, 11b, 11c and 11d) and the hinge region (13) or between the immunoglobulin Fc domain variant protein (12) and the biologically active protein. These fusion partners may be connected to the N-terminus or C-terminus of the immunoglobulin Fc domain variant protein (12) via the linker peptide (14). Figure 9-12 ).

[0102] In particular, the biologically active protein (11a) linked to the N-terminus of the fusion protein can be a ligand as a target moiety, and the other biologically active protein (11b) linked to the C-terminus of the same fusion protein can be a functional protein ( Figure 5 and 8). In contrast, the functional protein can be connected to the N-terminus, and the ligand can be connected to the C-terminus. These bispecific fusion proteins can form homodimers through intermolecular disulfide bonds via cysteine ​​residues present in the hinge region (13), and selective hydrophobic interactions between immunoglobulin Fc domain variant proteins can promote dimerization. Two different bispecific fusion proteins can form heterodimers. In this way, the homo- or heterodimeric protein according to an exemplary embodiment of the present invention can contain 1-4 biologically active proteins, and more types of proteins can be added by adding linker peptides to the biologically active proteins present at each end.

[0103] When aiming to prepare a heterodimer according to an exemplary embodiment of the present invention, in order to minimize the formation of homodimers, the knobs-into-holes (KIH) technique well known in the art may be used.

[0104] The term "knob-into-hole (KIH)" as used herein refers to a design strategy for heterodimerization of heavy chains in the production of bispecific IgG antibodies in antibody engineering. In the KIH technology, when two fusion proteins forming heterodimers are separated into a first fusion protein and a second fusion protein, in the modified IgG4 Fc region of the first fusion protein, serine as the 10th amino acid of the CH3 domain can be replaced by cysteine ​​(C), and threonine (T) as the 22nd amino acid can be replaced by tryptophan (W) (knob structure), and in the Fc region of the second fusion protein, tyrosine (Y) as the 5th amino acid of the CH3 domain can be replaced by cysteine ​​(C), threonine (T) as the 22nd amino acid can be replaced by serine (S), leucine (L) as the 24th amino acid can be replaced by alanine (A), and tyrosine (Y) as the 63rd amino acid can be replaced by valine (V) (hole structure). On the contrary, in the Fc region of the first fusion protein, tyrosine (Y) as the 5th amino acid of the CH3 domain can be replaced by cysteine ​​(C), threonine (T) as the 22nd amino acid can be replaced by serine (S), leucine (L) as the 24th amino acid can be replaced by alanine (A), and tyrosine (Y) as the 63rd amino acid can be replaced by valine (V) (hole structure), and in the modified IgG4 Fc region of the second fusion protein, serine (S) as the 10th amino acid of the CH3 domain can be replaced by cysteine ​​(C), and threonine (T) as the 22nd amino acid can be replaced by tryptophan (W) (knob structure).

[0105] In this case, the position of the amino acid in which the mutation occurs is based on the reference sequence (the amino acid sequence of the human IgG1 CH3 domain of SEQ ID NO: 77). Even when additional mutations occur at sites that are not dependent on the knob-into-hole structure on the CH domain (such as addition, deletion or substitution of amino acids), a fusion protein in which the amino acid corresponding to the position of the site based on the reference sequence is mutated can be used. Optionally, the knob-into-hole structure can be introduced by other amino acid mutations well known in the art. Such mutations are described in detail in previous literature (Wei et al., Oncotarget 8(31):51037-51049, 2017; Ridgway et al., Protein Eng. 9(7):617-621, 1996; Carter, P., J. Immunol. Methods 48(1-2):7-15, 2001; Merchant et al., Nat. Biotechnol. 16(7):677-681, 1998). The bispecific dimeric fusion protein can be produced by selective mutations such as a combination of a knob structure in which the threonine at the 22nd amino acid of the CH3 domain of the first fusion protein is substituted with tyrosine and a hole structure in which the tyrosine at the 63rd amino acid of the CH3 domain of the second fusion protein is substituted with threonine. Conversely, a knob-into-hole structure can be formed by introducing a hole structure into the first fusion protein and a knob structure into the second fusion protein.

[0106] According to still another aspect of the present invention, there is provided a composition containing a fusion protein, a polynucleotide encoding the fusion protein, or a homo- or hetero-dimer thereof as an active ingredient.

[0107] According to still another aspect of the present invention, there is provided a method for prolonging the in vivo half-life of a biologically active protein, the method comprising the step of administering to a subject a fusion protein obtained by fusing the biologically active protein to an immunoglobulin Fc domain variant protein, or a homo- or hetero-dimer containing the fusion protein.

[0108] According to still another aspect of the present invention, there is provided a composition containing, as an active ingredient, a fusion protein obtained by fusing a biologically active protein to an immunoglobulin Fc domain variant protein, or a homo- or hetero-dimer of the fusion protein.

[0109] The composition may contain a pharmaceutically acceptable carrier, and may further include a pharmaceutically acceptable adjuvant, excipient or diluent in addition to the carrier.

[0110] The term "pharmaceutically acceptable" as used herein refers to physiologically acceptable and does not usually cause allergic reactions (such as gastrointestinal disorders and dizziness) or compositions of similar reactions when given to the human race. The example of carrier, excipient and diluent includes lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic (acacia rubber), alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum, magnesium stearate and mineral oil. In addition, filler, anti-aggregating agent, lubricant, wetting agent, essence, emulsifier and preservative can be further contained.

[0111] In addition, when the pharmaceutical composition according to an exemplary embodiment of the present invention is administered to a mammal, the pharmaceutical composition can be formulated using methods known in the art to enable rapid, sustained or delayed release of the active ingredient. Examples of formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powder forms.

[0112] The composition according to an exemplary embodiment of the present invention can be given by various routes, such as oral administration and parenteral administration, for example, suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal or intraspinal administration, and can be given using an implant device for sustained release or continuous or repeated release. The administration can be performed once a day or several times within the desired range, and can be performed at intervals such as once a week, twice a week, and once a month, and the duration of administration is not particularly limited either.

[0113] The composition according to an exemplary embodiment of the present invention can be formulated with a commonly used pharmaceutically acceptable carrier in a suitable form. The example of a pharmaceutically acceptable carrier includes a carrier for parenteral administration, such as water, suitable oil, saline solution, glucose aqueous solution and glycol, and may further contain a stabilizer and a preservative. The example of a suitable stabilizer includes an antioxidant, such as sodium bisulfite, sodium sulfite or ascorbic acid. The example of a suitable preservative includes benzalkonium chloride, methyl or propyl paraben and chlorobutanol. In addition, if necessary, depending on the method of administration or preparation, the composition according to the present invention may suitably contain a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjusting agent, an analgesic agent, a buffer, an antioxidant, etc. Suitable for pharmaceutically acceptable carriers and preparations of the present invention, including those illustrated above, are described in detail in the document [Remington's Pharmaceutical Sciences, latest edition].

[0114] The dosage of the composition to the patient depends on many factors, including the patient's height, body surface area, age, specific compound to be administered, sex, time and route of administration, general health, and other drugs administered simultaneously. The pharmaceutically active protein can be administered in an amount of 100 ng / body weight (kg)-10 mg / body weight (kg), more preferably 1-500 μg / kg (body weight), and most preferably 5-50 μg / kg (body weight), but the dosage can be adjusted considering the above factors.

[0115] According to still another aspect of the present invention, a method for treating a disease in a subject is provided, the method comprising the step of administering to the subject a therapeutically effective amount of a fusion protein obtained by fusing a biologically active protein to an immunoglobulin Fc domain variant protein, or a homo- or hetero-dimer containing the fusion protein.

[0116] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined based on factors including the type, severity, age, sex, drug activity, drug sensitivity, administration time, administration route, excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The therapeutically effective amount of the composition of the present invention is 0.1 mg / kg-1 g / kg, and more preferably 1 mg / kg-500 mg / kg, but the effective dose can be appropriately adjusted according to the age, sex, and condition of the patient.

[0117] The above-mentioned diseases are diseases that require the administration of biologically active proteins, for example, when the biologically active protein is a GLP-1 analog, the disease to be treated may be diabetes or metabolic syndrome; when the biologically active protein is a GLP-2 analog, the disease to be treated may be short bowel syndrome or inflammatory bowel disease; when the biologically active protein is IL-2, the disease to be treated may be a malignant tumor; when the biologically active protein is FcεRIa, the disease to be treated may be an IgE-mediated allergic disease, such as asthma and atopic dermatitis; and when the biologically active protein is an anti-CD40L antibody mimetic, the disease to be treated may be an autoimmune disease or organ transplant rejection.

[0118] Hereinafter, the present invention will be described in more detail by examples and experimental examples. However, the present invention is not limited to the examples and experimental examples described below, and can be implemented in various other forms, and the examples and experimental examples described below are provided to make the disclosure of the present invention complete and to fully convey the scope of the present invention to the technicians in the field to which the present invention belongs.

[0119] Example 1: Design of immunoglobulin Fc domain variant protein

[0120] The technology for connecting the Fc domain of an antibody to a biologically active protein or an active pharmaceutical ingredient (API) to increase the half-life of the API is a widely used technology. However, when an N-linked sugar chain is added to asparagine, which is the 297th amino acid, in the Fc domain portion of an antibody, the sugar chain pattern is different from that of a human antibody, or it is inconsistent and produced in a heterogeneous form, depending on the characteristics of the host cell used for antibody production. Therefore, the activity of the fusion protein to which the API is connected may not be constant, or may exhibit unnecessary effects (such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)) in some cases, resulting in side effects. In addition, it is known that the reason for the increase in the half-life after in vivo administration of an antibody is that the antibody is absorbed due to endocytosis of vascular endothelial cells in vivo, and then binds to the neonatal Fc receptor (hereinafter referred to as "FcRn") due to the low pH in the endosomal state to avoid degradation by the lysosome, and separates from the FcRn in the process of endosomal recycling to the cell membrane under extracellular physiological pH conditions ( Fig.13 ). It is known that antibody recycling via FcRn exhibits an effect of extending the half-life of the antibody to as long as about 21 days (Raghavan, M. et al., Biochem, 34(45): 14649-14657, 1995).

[0121] However, since existing antibody Fc domains have different affinities with FcRn depending on the type of IgG, there is a limit to improving the half-life of the API.

[0122] Therefore, the present inventors designed an Fc domain protein variant that has no ADCC and CDC activities while increasing the affinity of the existing Fc domain protein for FcRn.

[0123] Therefore, the present inventors designed variants (SEQ ID NOs:6-9) in which threonine (T) as the 18th amino acid of the modified IgG4 Fc protein having the amino acid sequence shown in SEQ ID NOs:1-4 was substituted with glutamine (Q), and methionine (M) as the 196th amino acid was substituted with leucine (L), and the variant was named "NTIG (SEQ ID NO:5)".

[0124] Example 2: Preparation of monospecific fusion protein constructs

[0125] Subsequently, the present inventors prepared various fusion proteins in which various biologically active proteins were linked to the N-terminus and / or C-terminus of the immunoglobulin Fc domain variant protein (NTIG) prepared in Example 1.

[0126] 2-1: Design and production of GLP-1E-NTIG

[0127] The present inventors designed a fusion protein (GLP-1E-NTIG, SEQ ID NO: 50) in which the NTIG protein prepared in Example 1 (SEQ ID NO: 7) was linked to the C-terminus of a GLP-1 / exendin-4 hybrid peptide (SEQ ID NO: 49) which is a hybrid peptide of GLP-1 and exendin-4 through a linker peptide ((G4S)3-IgD / IgG1 hybrid hinge, SEQ ID NO: 14) containing a hinge region in a hybrid form, and the fusion protein was named "PG-11". GLP-1E-NTIG was designed to add a tPA signal sequence (SEQ ID NO: 51) for secretion from a host cell. At the same time, a polynucleotide encoding PG-11 was synthesized using oligonucleotide synthesis, PCR amplification, and site-directed mutagenesis techniques, and then inserted into a pGP30 expression vector (Genexine, Inc., Korea). The prepared vector was transiently expressed using an ExpiCHO kit manufactured by Thermo Fisher Scientific Solutions LLC. Specifically, the vector prepared as above and the ExpiFectamine reagent contained in the kit were mixed in ExpiCHO-S cells, and the resultant was cultured in an incubator under conditions of 8% CO2 and 37°C for 1 day, then the temperature was lowered to 32°C and cultured until the 7th day.

[0128] Afterwards, protein A capture and purification were performed, and the candidate material was checked for purification by SDS-PAGE analysis under non-reducing and reducing conditions, and formulated with formulation buffer according to the pI value of the candidate material. The formulated material was quantified using Nano drop, and the final purity was checked using SEC-HPLC ( Fig.14a and 14b ).

[0129] 2-2: Design and production of GLP-2-2G-NTIG

[0130] The present inventors designed a fusion protein (GLP-2-2G-NTIG, SEQ ID NO: 53) in which NTIG (SEQ ID NO: 7) was linked to the C-terminus of a GLP-2 analog (GLP-2-2G, SEQ ID NO: 52) via a linker peptide (SEQ ID NO: 14), the GLP-2 analog being a mutant in which alanine (A) as the second amino acid of the human GLP-2 peptide was substituted with glycine (G), and the fusion protein was named "PG-22". Similarly, PG-22 was also designed to add a tPA signal sequence for secretion (SEQ ID NO: 51). Similar to Example 2-1, a polynucleotide encoding PG-22 was prepared and inserted into a pGP30 expression vector (Genexine, Inc., Korea) to produce the protein using the ExpiCHO system, and then the final purity was checked ( Fig.15a and 15b ).

[0131] 2-3: Design and production of NTIG-IL-10V

[0132] The present inventors designed a fusion protein (NTIG-IL-10V, SEQ ID NO: 55) in which an IL-10 variant (SEQ ID NO: 54) was linked to the C-terminus of NTIG via a linker peptide (SEQ ID NO: 26), and the fusion protein was named "PG-088". Similar to Example 2-1, a polynucleotide encoding PG-088 was prepared and inserted into a pBispec expression vector (Genexine, Inc., Korea) to produce the protein using the ExpiCHO system, and then the final purity ( Fig.16a and 16b ).

[0133] 2-4: Design and production of NTIG-IL-10V monomer

[0134] The present inventors designed a fusion protein (NTIG-IL-10Vm, SEQ ID NO: 57) in which the IL-10 monomer variant (SEQ ID NO: 56) was linked to the C-terminus of NTIG via a linker peptide (SEQ ID NO: 26), and the fusion protein was named "PG-088m". Similar to Example 2-1, a polynucleotide encoding PG-088m was prepared and inserted into the pBispec expression vector to produce the protein using the ExpiCHO system, and then the final purity was checked ( Fig.17a and 17b). The IL-10 monomer variant (IL-10Vm) is obtained by introducing a mutation for selectively inhibiting the immunostimulatory activity of the IL-10 protein (having both different activities of immunostimulatory activity and immunosuppressive activity), and the IL-10 monomer variant is a monomer variant protein produced in a monomeric form by inserting a linker of GGSGGSGGS (SEQ ID NO: 46) in the middle of the protein production process to solve the problem of aggregates generated due to self-dimerization in the process.

[0135] Example 3: Preparation of bispecific fusion protein gene constructs

[0136] 3-1: Preparation of anti-PD-L1-NTIG-IL-2

[0137] A polynucleotide encoding each of a fusion protein in which a scFv targeting PD-L1 was linked to the N-terminus of NTIG (SEQ ID NO: 7) and IL-2 was linked to the C-terminus (anti-PD-L1-NTIG-IL-2) and a fusion protein in which a modified IgG4 Fc domain protein of SEQ ID NO: 2 was used instead of NTIG (anti-PD-L1 modified IgG4 Fc-IL-2) was inserted into the pBispec expression vector, and each protein was produced using the ExpiCHO system.

[0138] 3-2: Design and production of FcεRIα-NTIG-IL-10V

[0139] The present inventors designed a bispecific fusion protein (FcεRIα-NTIG-IL-10V, SEQ ID NO: 59), in which the extracellular domain of FcεRIα as a receptor specifically binding to IgE (SEQ ID NO: 58) was connected to the N-terminus of NTIG-IL-10V designed in Example 2-3 through a linker peptide (SEQID NO: 18), and the bispecific fusion protein was named "PG-075", and a polynucleotide encoding the fusion protein was prepared and inserted into the pBispec expression vector. The PG-075 construct was also designed so that the tPA signal sequence (SEQ ID NO: 51) was located at the N-terminus for secretion, and the construct was produced using the ExpiCHO system, and subjected to SDS-PAGE and western blotting ( Fig.18a and 18b ).

[0140] 3-3: Design and production of FcεRIα-NTIG-IL-10Vm

[0141] The present inventors designed a bispecific fusion protein (FcεRIα-NTIG-IL-10Vm, SEQ ID NO: 60), in which the extracellular domain of FcεRIα as a receptor that specifically binds to IgE (SEQ ID NO: 58) is connected to the N-terminus of NTIG-IL-10Vm designed in Example 2-4 through a linker peptide (SEQID NO: 18), and the bispecific fusion protein is named "PG-110", and a polynucleotide encoding the fusion protein is prepared and inserted into the pAD15 expression vector. The PG-110 construct is also designed so that the tPA signal sequence (SEQ ID NO: 51) is located at the N-terminus for secretion. The gene inserted into the pAD15 expression vector is transfected into CHO DG44 cells, followed by methotrexate (MTX) amplification, and a monoclonal sorting operation is performed to prepare a high-efficiency expression cell line, and then the PG-110 protein is produced and purified from the high-efficiency expression cell line ( Fig.19a and 19b ).

[0142] Example 4: Preparation of anti-CD40L hybrid antibody

[0143] 4-1: Preparation of chimeric anti-CD40L antibodies

[0144] The present inventors prepared an anti-CD40L antibody obtained by introducing an FcγRIIα binding inhibitory variant into the heavy chain CH2 and CH3 domains of an antibody (C10) in which the Fc region of the mouse-derived CD40L (anti-CD154) antibody described in WO2016 / 182335A1 was replaced with a human IgG1 Fc region, and the anti-CD40L antibody was named "C10M".

[0145] 4-2: Preparation of chimeric anti-CD40L-NTIG hybrid antibody

[0146] The present inventors designed a hybrid heavy chain protein in which the heavy chain portion in the Fab portion of the C10M antibody was linked to the N-terminus of the NTIG protein prepared in Example 1 that does not bind to FcγR, and the hybrid heavy chain protein was named "PG-129".

[0147] The heavy chain portion (V H -CH1, SEQ ID NO: 61) connected to the hybrid heavy chain of NTIG (SEQ ID NO: 7) and inserted into the pAD15 expression vector (WO2015 / 009052A). In addition, the light and heavy parts (V L -CL , SEQ ID NO: 62) was inserted into the pAD15 expression vector so that the polynucleotide could be regulated under a dual promoter. The vector was then co-transfected into CHO DG44 (from Dr. Chasin, Columbia University, USA) cells using the Neon transfection system.

[0148] The expression vector completed as above was also transiently expressed using an ExpiCHO kit manufactured by Thermo Fisher Scientific Solutions LLC. Specifically, the vector construct prepared as above and the ExpiFectamine reagent contained in the kit were mixed in ExpiCHO-S cells, and the resultant was cultured in an incubator under conditions of 8% CO2 and 37°C for 1 day, then the temperature was lowered to 32°C, and cultured until the 7th day.

[0149] Afterwards, protein A capture and purification were performed, and the candidate material was checked for purification by PAGE analysis and western blot analysis under non-reducing and reducing conditions, and formulated with formulation buffer according to the pI value of the candidate material. The formulated material was quantified using Nano drop, and the final purity was checked using SEC-HPLC.

[0150] As a result, Fig.20a and 20b As shown, the light chain and the heavy chain were normally expressed, a single band appeared under non-reducing conditions, and thus it could be found that normal heterotetramers were formed. In addition, as a result of SEC-HPLC analysis, the purity was 97.9%, which was very high.

[0151] 4-3: Preparation of humanized anti-CD40L-NTIG hybrid antibody

[0152] Chimeric antibodies have substantially improved the shortcomings of conventional human antibodies by introducing the NTIG technology (immunoglobulin Fc domain variant protein) of the present invention, but the inventors have determined that the antigen binding site is still that of a mouse-derived antibody, and therefore an unnecessary immune response may occur in the human body, and in order to prepare a safer hybrid antibody, a humanized antibody (anti-CD40LFab-NTIG) was designed, in which the framework portion other than the antigenic determinant of the Fab of the antigen-binding fragment of the antibody as the basis of the present invention was replaced by the sequence of a human antibody (Tables 1 and 2), and the humanized antibody was named "PG-400".

[0153] In addition, the present inventors designed and prepared a bispecific hybrid antibody (anti-CD40L Fab-NTIG-IL-10Vm), in which an IL-10 monomer variant protein (SEQ ID NO: 56) as a cytokine with immunosuppressive activity was linked to the heavy chain C-terminus of the hybrid antibody (Table 3), and the bispecific hybrid antibody was named "PG-410". The light chain structure of the bispecific hybrid antibody is the same as shown in Table 2.

[0154] [Table 1] Structure of the heavy chain of humanized anti-CD40L-NTIG hybrid antibody

[0155]

[0156] [Table 2] Structure of the light chain of the humanized anti-CD40L-NTIG hybrid antibody

[0157]

[0158] [Table 3] Structure of the heavy chain of the bispecific anti-CD40L-NTIG hybrid antibody

[0159]

[0160] To produce humanized anti-CD40L hybrid antibodies (PG-400 and PG-410), polynucleotides encoding each of the heavy and light chains of the antibodies were prepared, and then for transient expression in animal cells, plasmid DNA was transfected into HEK293F cells using the N293F vector system (Y-BIOLOGICS). To prepare nucleic acids, 25 μg of plasmid DNA was added to 3 ml of culture medium and mixed, and then 25 μl of 2 mg / ml of polyethyleneimine (PEI, PolyPlus, USA) was added thereto, followed by mixing. The reaction solution was allowed to stand at room temperature for 15 minutes, and then added to a 1×10 6 The cells were cultured in 40 ml of a culture medium containing 10 cells / ml, and the resultant was cultured for 24 hours at 120 rpm, 37° C., and 8% CO 2 . 24 hours after DNA transfection, a nutrient supplement medium component (Soytone, BD, USA) was added to a final concentration of 10 g / L. After 7 days of culture, the cell culture medium was centrifuged at 5000 rpm for 10 minutes to collect the supernatant.

[0161] Subsequently, the protein A resin was filled into the column and washed with 1×DPBS, the collected supernatant was bound to the resin at a rate of 0.5 ml / min at 4°C, and the protein was eluted with 0.1 M glycine. To replace the buffer, the eluted solution was added to a dialysis tube (GeBAflex tube, Geba, Israel) and dialyzed with 1×DPBS at 4°C. The resulting material was gel filtered using Superdex 200 resin and then formulated with PBS (pH 7.4) buffer.

[0162] For the purified hybrid antibodies, the yield and purity of the hybrid antibodies were determined by SDS-PAGE analysis, size exclusion FPLC and size exclusion HPLC analysis.

[0163] As a result, Fig.21a , 21b As shown in Figures 22a and 22b, it was found that PG-400 and PG-410 to which the humanized anti-CD40L hybrid antibody of the present invention was applied were produced at high purity.

[0164] 4-4: Design of anti-CD40L scFv-NTIG-IL-10 fusion protein

[0165] The present inventors sorted out the variable region portion of the Fab of the humanized anti-CD40L antibody, designed scFv using the sorted variable region portion, and inserted the scFv to replace the V of the heavy chain construct of the anti-CD40L antibody used in Example 4-3. H A single-chain antibody-binding fragment fusion protein was designed based on the -CH1 portion, and a bispecific fusion protein in which the IL-10 monomer variant (SEQ ID NO: 56) used in Example 4-3 was connected to the C-terminus of the anti-CD40L scFv-NTIG via a linker peptide (SEQ ID NO: 26) was designed (Table 4). In this example, the anti-CD40L scFv is composed of the light chain variable region (V L ), linker, heavy chain variable region (V H ), but on the contrary, it can be composed of V H , connector and V L Since the CD40L-binding ability is maintained even in the case of using the latter anti-CD40L scFv, and regarding the type of linker, various types can be used.

[0166] [Table 4] Structure of anti-CD40L scFv-NTIG-IL-10 fusion protein

[0167]

[0168] Example 5: Preparation of heterodimers

[0169] The present inventors designed bispecific fusion proteins containing both GLP-1 analogs and GLP-2 analogs, as shown in Table 5. In these bispecific fusion proteins, a knob-into-hole (KIH) structure was introduced to form heterodimers through intermolecular disulfide bonds and hydrophobic interactions between the first fusion protein and the second fusion protein.

[0170] The difference between Examples 5-1 and 5-2 is whether an N-linked sugar chain attachment site is included so that an N-glycan sugar chain can be attached to the linker that connects the GLP-1 analog of the first fusion protein to NTIG. This is because GLP-1, as well as exendin 4 (Ex4), oxyntomodulin (OXM), and GLP-2 all bind to GLP-1R, which is a GLP-1 receptor, while only GLP-2 binds to GLP-2R, and GLP-1R is expressed in various organs (such as the brain, heart, liver, muscle, and pancreas) in addition to the gastrointestinal tract (GI tract), and therefore it is necessary to slightly reduce the affinity with GLP-1R so that GLP-1 can specifically act in the gastrointestinal tract. For this reason, in order to slightly reduce the binding force of the GLP-1 analog to GLP-1R, the present inventors used a protein to which a glycan attachment domain was added so that glycans could be attached to the linker portion (including the IgD / IgG1 hybrid hinge) of the GLP-1 analog (Example 5-1).

[0171] [Table 5] Design of fusion protein for forming GLP-1 / GLP-2 heterodimer according to an exemplary embodiment of the present invention

[0172]

[0173] The vector construct prepared as above was transiently expressed using an ExpiCHO kit manufactured by Thermo Fisher Scientific Solutions LLC. Specifically, the vector construct prepared as above and the ExpiFectamine reagent contained in the kit were mixed in ExpiCHO-S cells, and the resultant was cultured in an incubator under conditions of 8% CO2 and 37°C for 1 day, then the temperature was lowered to 32°C, and cultured until the 7th day.

[0174] The supernatant obtained by the culture and the heterodimeric proteins of Examples 5-1 and 5-2 purified by a protein A column and a secondary column (named 'MG12-4' and 'MG12-5', respectively) were appropriately diluted with 4X LDS sample buffer and water for injection to prepare samples so that the final concentration was 3-10μg / 20μL. In the case of samples under reducing conditions, each substance to be analyzed, 4X LDS sample buffer, 10X reducing agent and water for injection were appropriately diluted to prepare samples so that the final concentration was 3-10μg / 20μL, and the samples were heated in a 70°C heating block for 10 minutes. The prepared sample was loaded in an amount of 20μL into each well of the gel fixed in the pre-installed electrophoresis device. For size marking, the sample was loaded at 3-5μL / well. Electrophoresis was performed after the power supply was set to 120V and 90 minutes. The gel subjected to electrophoresis was separated and then stained with a staining solution and a decolorizing solution, and the results were analyzed. As a result, as Fig.23 As shown, it can be confirmed that the heterodimer according to an exemplary embodiment of the present invention is normally produced.

[0175] Example 6: Analysis of the influence of hinges on the production of fusion proteins

[0176] The present inventors studied how the production efficiency of the protein varies depending on the type of hinge when an API is linked to the N-terminus of the NTIG protein while linking various types of API to the N-terminus and / or C-terminus of the NTIG protein of the present invention.

[0177] 6-1: Preparation of FcεRIα-NTIG-IL-13α2

[0178] The present inventors designed a bispecific fusion protein (FcεRIα-NTIG-IL-13Rα2) in which FcεRIα (SEQID NO: 58) was linked to the N-terminus of NTIG (SEQ ID NO: 7) and IL-13Rα2 (SEQ ID NO: 74) was linked to the C-terminus, and the bispecific fusion protein was named "PG-50". In producing PG-50, the protein productivity of linking FcεRIα to the hinge region of NTIG was studied when using an IgD hinge (SEQID NO: 15) or a combination of a (G4S)3 linker and an IgG1 hinge (SEQ ID NO: 16). Thus, the fusion protein using the IgD hinge was named "PG-50-1", and the fusion protein using the (G4S)3 linker / IgG1 hinge was named "PG-50-2". In the case of the IgD hinge, one intermolecular disulfide bond is formed, whereas in the case of the (G4S)3 linker / IgG1 hinge, two intermolecular disulfide bonds are formed.

[0179] As a result, Fig.24a and 24b As shown, it was found that the yield of the target material of the fusion protein containing the IgG1 hinge (PG-50-2) was increased by about 15% compared with that of PG-50-1 containing the IgD hinge.

[0180] 6-2: Preparation of GLP-2-2G-NTIG-IL-10wt

[0181] The inventors analyzed the effects of various types of hinges on protein yield using different types of APIs. For analysis, specifically, a construct in which GLP-2-2G (SEQ ID NO: 52) was connected to the N-terminus of NTIG (SEQ ID NO: 7) and IL-10wt (SEQ ID NO: 75) was connected to the C-terminus of NTIG was prepared. Here, the bispecific fusion protein (GLP-2-2G-NTIG-IL-10wt) was designed by connecting GLP-2-2G to NTIG using a (G4S)3 linker + IgD / IgG1 hybrid hinge (SEQ ID NO: 14), and connecting IL-10wt to the C-terminus of NTIG using a linker peptide consisting of the amino acid sequence shown in SEQ ID NO: 26, and was named "PG-073". A polynucleotide encoding the fusion protein designed as above was prepared and then inserted into the pBispec expression vector, the fusion protein was expressed using the ExpiCHO system according to the method used in Example 2-1, and then the protein expression level in the culture supernatant was examined by SDS-PAGE. The IgD / IgG1 hybrid hinge forms two intermolecular disulfide bonds.

[0182] As a result, Fig.24c As shown, in the case of PG-073, the size of the main band under non-reducing conditions was 110 kDa, and it was confirmed that a bispecific fusion protein connecting both GLP-2-2G and IL-10 was formed. However, a band in which one or both of GLP-2 was cut was found below the main band. Therefore, it was found that the (G4S)3 linker + IgD / IgG1 hybrid hinge linker used between GLP-2 and NTIG was slightly unstable. In addition, the amount of fusion protein produced based on 100 ml of culture solution was only 0.5 mg (PG-073), and thus the protein productivity was reduced, and the purity was also about 66%, which was low.

[0183] 6-3 and 6-4: Preparation of GLP-2-2G (3 spots)-NTIG-IL-10Vm

[0184] Therefore, the present inventors designed a fusion protein (GLP-2V (3 points) -NTIG-IL-10Vm), wherein in the GLP-2-2G-NTIG-IL-10wt fusion protein, GLP-2-2G was changed into a mutant containing 3 point mutations (SEQ ID NO: 76), the hinge part was replaced with a different type of linker or hinge (SEQ ID NO: 17 or 18 respectively), and IL-10wt was replaced with IL-10Vm prepared in Example 2-4, and the fusion proteins were named "PG210-5 (Example 6-3)" and "PG210-6 (Example 6-4)", respectively.

[0185] After the polynucleotide encoding each fusion protein was inserted into the pBispec expression vector, the fusion protein was expressed using the ExpiCHO system according to the method used in Example 2-1, and then the protein expression level in the culture supernatant was examined by SDS-PAGE.

[0186] As a result, Fig.24d and 24e As shown, in the case of PG-210-5, 3.48 mg of protein was found based on 200 ml of culture solution and the purity was found to be about 88.3%, and in the case of PG-210-6, 5 mg of protein was found and the purity was found to be 90.7%.

[0187] Experimental Example 1: Analysis of FcRn Binding Affinity

[0188] The present inventors compared the binding affinity of the NTIG protein of the present invention to FcRn with that of IgG4 Fc and IgG1 antibodies.

[0189] For comparison, specifically, recombinant FcRn (rhFcRn, R&D systems, USA) as a ligand was dissolved in an acetate buffer at a concentration of 5 μg / ml, and then immobilized on a biosensor chip (Series SCM5 sensor chip GE Healthcare, USA) using amine coupling.

[0190] Subsequently, in order to compare the FcRn binding affinity of the NTIG of the present invention with the FcRn binding affinity of IgG4 Fc, the present inventors analyzed the binding affinity of PG-11 and dulaglutide (trade name dulaglutide, which is a fusion protein in which commercial GLP-1 is linked to IgG4 Fc) prepared in Example 2-1 to FcRn by surface plasmon resonance (SPR). As an analyzer, Biocore 8K (GE Healthcare, USA) was used, and all reagents used in the analysis were purchased from GE Healthcare. PG-11 and dulaglutide were dissolved in running buffer (PBST, pH 6.0), and then serially diluted from 1,000 nM to 1 / 2, and multi-cycle kinetic analysis was performed. The experiment was performed at 25°C at a flow rate of 30 μl / min, and the association of the analyte was performed for 60 seconds, and then the dissociation was performed for 60 seconds. Based on the experimental results, the optimal antibody concentrations (Dubotran: 8,000 nM and PG-11: 2,000 nM) were selected, multi-cycle kinetic analysis was performed, and the equilibrium dissociation constant (K) was measured using the Biacore Insight program (GE Healthcare, USA). D ). At this time, the buffer was replaced with PBS buffer at pH 7.4 during regeneration.

[0191] As shown in Table 6 and Fig.25 and 26 As shown, the steady-state affinity K D The results of comparative analysis show that the K D The K value is 2,090nM and PG-11 D The value was 199 nM, and the results showed that the affinity of PG-11 according to an exemplary embodiment of the present invention to FcRn was about 5.3 times higher than that of Duda. It is known that the binding affinity to FcRn is related to the pharmacokinetics of Fc fusion proteins, and therefore, the in vivo half-life of PG-11 is expected to be longer than that of Duda (Mackness et al., MAbs. 11(7): 1276-1288, 2019).

[0192] [Table 6] Affinity analysis results of PG-11 and DuyiDa with FcRn

[0193] Analytes PG-11 Du Yi Da <![CDATA[Steady-state affinity (K D )]]> 397±19.3nM 2,090±75.5nM <![CDATA[CV%(Steady-state affinity, K D )]]> 4.86 3.61

[0194] In addition, in order to compare the FcRn binding affinity of the NTIG of the present invention with that of IgG4 Fc, the present inventors analyzed the binding affinity of PG-11 and rituximab, a commercial recombinant chimeric anti-CD20 antibody of the IgG1 family, to FcRn by surface plasmon resonance (SPR). PG-11 and rituximab were dissolved in PBST running buffer (pH 6.0) at various concentrations (2,000 nM-1.96 nM for PG-11 and 4,000 nM-3.9 nM for rituximab), and then similar to the comparative analysis of PG-11 and DuliDa, association, dissociation and regeneration were performed under the conditions of association for 60 seconds, dissociation for 60 seconds and regeneration for 30 seconds. During regeneration, a PBS buffer with a pH of 7.4 was used.

[0195] As shown in Table 7 and Fig. 27 As shown, the steady-state affinity K D The results of the comparative analysis showed that the K D The K value is 2,390nM and PG-11 D The value was 256 nM, and the results indicated that the affinity of PG-11 according to an exemplary embodiment of the present invention to FcRn was about 9.3 times higher than that of rituximab.

[0196] [Table 7] Results of affinity analysis of PG-11 and rituximab for FcRn

[0197] Analytes PG-11 Rituximab <![CDATA[Steady-state affinity (K D )]]> 256nM 2,390±242nM <![CDATA[CV% steady-state affinity (K D )]]> - 10.15

[0198] As described above, NTIG according to an exemplary embodiment of the present invention has high affinity to FcRn compared to the Fc region of conventional IgG1 or IgG4, and thus when NTIG is administered in vivo, the half-life is expected to be significantly improved.

[0199] Subsequently, the present inventors aimed to compare the affinity of the modified IgG4 Fc domain protein of SEQ ID NO: 2 to FcRn based on NTIG (SEQ ID NO: 7) according to an exemplary embodiment of the present invention.

[0200] For comparison, the inventors did not calculate a simple K D Instead, the steady-state affinity (K) was calculated by associating the protein at a pH of 6.0 and dissociating at a pH of 7.3. D) (Piche-Nicholas et al., MAbs. 10(1):81-94, 2018). Specifically, for the binding affinity of anti-PD-L1-NTIG-IL-2 and anti-PD-L1 modified IgG4Fc-IL-2 prepared in Example 3-1 to rhFcRn, SPR analysis was performed by changing the pH conditions during association and dissociation as described above.

[0201] As a result, as shown in Table 8 and Fig.28 As shown, it was found that the affinity of anti-PD-L1-NTIG-IL-2 to FcRn using NTIG tended to be higher than that of anti-PD-L1-modified IgG4 Fc-IL-2 using modified IgG4 Fc. However, a large amount of protein aggregates were generated in the anti-PD-L1-modified IgG4 Fc-IL-2 protein sample during the exchange of the binding buffer, and thus there was a problem of poor reliability of the results. Therefore, the same experiment was performed again by changing the test material to the ligand and FcRn to the sample.

[0202] These results indicate that the use of NTIG exhibits an effect of extending the in vivo half-life by enhancing the FcRn binding ability of the produced protein as well as superior effects on the physical properties and stability of the protein, compared to the previously known use of the modified IgG4 Fc of SEQ ID NO: 2.

[0203] [Table 8] Affinity analysis results of NTIG and modified IgG4 Fc to FcRn

[0204]

[0205] As buffers used for analysis, running buffer (1X PBST, pH 7.3), sample dilution buffer (1X PBST, pH 6.0), dissociation buffer (1X PBST, pH 7.3), and regeneration buffer (1X PBS, pH 8.0) were used.

[0206] As the analysis results, as shown in Table 9 and Fig.29As shown, it was found that both proteins exhibited rapid binding and rapid dissociation patterns, and both proteins tended to dissociate well from binding to FcRn under neutral conditions (pH 7.3), but under pH 6.0 conditions, the affinity of the anti-PD-L1-NTIG-IL-2 protein was 5.06 times higher than that of the anti-PD-L1-modified IgG4 Fc-IL-2 protein. Therefore, compared with the anti-PD-L1-modified IgG4 Fc-IL-2 protein, the anti-PD-L1-NTIG-IL-2 protein showed a tendency to rapidly bind to FcRn even at a lower concentration under pH 6.0 conditions. Considering that Fc-containing proteins taken up into cells by endocytosis bind to FcRn under the low pH conditions of endosomes, this result strongly suggests that fusion proteins containing NTIG will be more efficiently recycled by FcRn.

[0207] [Table 9] Affinity analysis results of NTIG and modified IgG4 Fc to FcRn (conversion between ligand and analyte)

[0208]

[0209] Experimental Example 2: Analysis of Binding Affinity to Various Fcγ Receptors It is known that the Fc region of IgG1 has binding affinity to FcγRI and FcγRIIIa. These Fc receptors are associated with effector functions (such as ADCC and CDC), and contribute to anticancer therapy when using antibodies or Fc fusion proteins for anticancer therapy, but have the disadvantage of causing side effects when using APIs for the treatment of diseases other than anticancer therapy.

[0210] Therefore, the present inventors analyzed the extent of affinity of the NTIG fusion protein according to an exemplary embodiment of the present invention to FcγRI and FcγRIIIa compared to IgG1 antibody by biolayer interferometry (BLI).

[0211] As a result, Fig.30 As shown, rituximab, which is an IgG1-based antibody, was found to specifically bind to FcγRI and FcγRIIIa, whereas PG-11, which is a fusion protein containing NTIG according to an exemplary embodiment of the present invention, did not specifically bind to these antibody receptors.

[0212] Furthermore, the present inventors conducted a comparative analysis of the binding affinities of rituximab and PG-11 according to an exemplary embodiment of the present invention to FcγRIIa, FcγRIIb, and FcγRIIIb.

[0213] As a result, Fig.31As shown, rituximab was found to specifically bind to three Fc receptors. Meanwhile, PG-11 according to an exemplary embodiment of the present invention showed weak binding, but considering the shape of the sensorgram, the binding was considered to be nonspecific binding.

[0214] Experimental Example 3: Analysis of Binding to Complement C1q

[0215] The antibody Fc region can induce complement-dependent cytotoxicity (CDC) by binding to complement C1q, and thus may cause serious problems in the clinical application of Fc fusion proteins for the treatment of general diseases other than cancer treatment. Therefore, the degree of binding affinity of a fusion protein containing NTIG according to an exemplary embodiment of the present invention to complement C1q was analyzed compared with rituximab, which is an IgG1-based antibody.

[0216] For comparison, specifically, PG-11 and rituximab were each diluted with coating buffer (0.1M Na2HPO4, pH 9.6) so that the concentration of each was 2 μg / ml. Subsequently, 100 μl of each diluted sample was loaded into each well and reacted overnight at 4°C. Next, 300 μl of washing buffer was added to the plate for washing 3 times each time, 200 μl of blocking buffer (2% BSA / PBS) was added to each well and reacted at room temperature for 2 hours. 300 μl of washing buffer was added to the plate, and then the washing was repeated 3 times, and the anti-C1q antibody was diluted to 10, 3, 1, 0.3 and 0.1 μg / ml with assay buffer at 1 / 3, and mixed (repeatedly) in an amount of 50 μl in each well, and then the resultant was reacted at room temperature for 2 hours. Then, the washing buffer was mixed in the plate at 300 μl each time, and the washing was repeated 8 times. The C1q-HRP conjugate was diluted 400 times, and 50 μl of the resultant was added to each well and reacted at room temperature for 1 hour. Washing buffer was added to the plate at 300 μl each time, and the washing was repeated 8 times, 50 μl of TMB as a substrate was mixed, and the resultant was reacted at room temperature. When the color reaction was blue, 50 μl of stop solution was added to each well to stop the reaction, and then the optical density was measured using a microplate reader set at 450 nm.

[0217] As a result of the analysis, Fig.32 As shown, it was found that rituximab bound to Clq in a concentration-dependent manner, whereas PG-11 according to an exemplary embodiment of the present invention did not bind to Clq.

[0218] Therefore, it was confirmed that NTIG according to an exemplary embodiment of the present invention is a safe protein carrier that can minimize side effects (such as ADCC or CDC) due to the Fc region while improving the half-life of API.

[0219] Experimental Example 4: Activity analysis of GLP-2

[0220] By cAMP Hunter TM The eXpress GPCR assay investigated whether many of the GLP-2-containing fusion proteins in the above examples appropriately exhibited GLP-2 activity (Table 10).

[0221] [Table 10] Constructs used for GLP-2 activity analysis

[0222] Example Code Construct structure 2-2 PG-22 GLP-2G-NTIG 5-1 MG12-4 GLP-1E / GLP-2G-NTIG (GLP-1w / Glycan Linker) 5-2 MG12-5 GLP-1E / GLP-2G-NTIG (GLP-1w / non-glycan linker) 6-3 PG-210-5 GLP-2V (3 points)-NTIG-IL-10Vm (patent connector) 6-4 PG-210-6 GLP-2V (3 points)-NTIG-IL-10Vm (GS+IgG1 hinge linker)

[0223] As a result of the analysis, Fig.33 As shown in Table 11, it was found that PG-210-6 in the fusion protein prepared according to one embodiment of the present invention exhibited a GLP-2 activity almost similar to that of PG-22 as a comparative example, and that PG-210-5 of another embodiment of the present invention exhibited an activity of about half that of PG-210-6. MG12-4 and MG12-5, which are heterodimers, were also found to exhibit a GLP-2 activity of only about half that of PG-22, but since MG12-4 and MG12-5 each contain one GLP-2 molecule, it can be predicted that in the case of MG12-4 and MG12-5, the activity is reduced by 50% compared to PG-22 in which two GLP-2 molecules are bound. PG-210-5 is a protein to which the linker peptide used in the previous patent document (US7,812,121B2) is applied, and it was found that the activity of PG-210-6 using the mixed linker peptide of (G4S)3+IgG1 hinge used in the present invention is very good.

[0224] [Table 11] Results of GLP-2 activity analysis

[0225] Example 2-2 5-1 5-2 6-3 6-4 <![CDATA[First EC 50 (nM)]]> 2.84 5.81 5.49 7.44 2.46 First relative activity (%) 100％ 48.8％ 51.7％ 38.1％ 115％ <![CDATA[Second EC 50 (nM)]]> 2.65 6.08 4.85 5.95 2.93 Second relative activity (%) 100％ 43.5％ 54.6％ 44.5％ 90％ Average activity (%) 100％ 46.15％ 53.15％ 41％ 102.5％

[0226] Experimental Example 5: Analysis of immunosuppressive activity of IL-10Vm fusion protein

[0227] 5-1: Affinity analysis for IL-10 receptor

[0228] The present inventors analyzed the affinity of the fusion proteins of Examples 2-3 and 2-4 to IL-10 receptor 1 (IL-10R1) by biolayer interferometry (BLI).

[0229] For analysis, the inventors used Dip and Read TMThe amine-reactive second generation (AR2G) kit (forteBio, catalog number 18-5092) attaches the IL-10R His-tagged protein to a 96-well plate. Specifically, 200 μl of D.W. was dispensed into a 96-well plate and inserted into the amine biosensor included in the kit for 10 minutes of hydration. Subsequently, after 200 μl of DW was dispensed into a new 96-well plate, EDC and NHS were mixed 1:1 in an amount of 1 / 20 of the required sample amount, and then diluted with DW, and 200 μl of the resultant was dispensed into a 96-well plate. Next, the IL-10R His-tagged protein was diluted to 10 μg / ml in a 10 mM acetate solution at pH 5.0, and then 200 μl of the resultant was added to the 96-well plate. After 200 μl of 1 M ethanolamine was added to the 96-well plate, the biosensor plate and the sample plate were placed in a 96-well plate. The instrument was placed in the instrument and the measurement was performed. After the measurement was completed, 200 μl of 1× kinetic buffer was added to the sample plate, and then the baseline was determined. Thereafter, the prepared NTIG-IL-10 fusion protein was diluted to various concentrations (0, 62.5, 125, 250, 500, and 1,000 nM) with 1× kinetic buffer, 200 μl of the resultant was dispensed to the sample plate, and then the The instrument measures Biolayer Interferometry (BLI).

[0230] [Table 12]

[0231] parameter Example 2-3 Embodiment 2-4 <![CDATA[K D (nM)]]> 11.1±0.9 29.2±8.85 <![CDATA[K on (1 / Ms)]]> 71100±6670 20550±2312 <![CDATA[K dis (1 / s)]]> 0.0008 0.0006 <![CDATA[R 2 ]]> 0.97 0.97

[0232] As a result, Fig.34 As shown in Table 12, the K of the fusion protein (NTIG-IL-10Vm) according to Example 2-4 of the present invention D The value is 29.2 nM, which is the K of the dimeric IL-10 fusion protein of Example 2-3. D The value of 11.1 nM was about 3 times higher than that of 11.1 nM. This result can be interpreted as the affinity of the monomeric IL-10 variant fusion protein to the receptor is lower than that of the dimeric IL-10 protein, but it can be predicted that the affinity will decrease due to monomer formation.

[0233] 5-2: Analysis of immunosuppressive activity of IL-10Vm fusion protein

[0234] The present inventors analyzed the in vitro immunosuppressive activity of various fusion proteins (PG-088m, PG-110, PG-410 and PG-210-6) containing a monomeric IL-10 variant protein (IL-10Vm) according to an exemplary embodiment of the present invention.

[0235] For the analysis, specifically, after removing the supernatant from a T75 flask containing cultured macrophages Raw264.7, washing was performed once with PBS, and then 3 ml of TrypLE TM Select enzyme and react the result at 37°C for 3-5 minutes. After that, add 10 ml of fresh medium to the T75 flask, transfer the cell suspension from the bottom of the flask to a 50 ml conical tube, and then centrifuge at 1,500 rpm for 5 minutes. Next, remove the supernatant and tap the test tube gently to release the cells. Then, mix 5 ml of fresh medium and count the cells. Adjust the cell concentration to 1×10 5 cells / ml, and 100 μl of the resultant was dispensed into a 96-well flat-bottom plate.

[0236] After that, PG-088m prepared in Example 2-4, PG-110 prepared in Example 3-3, PG-410 prepared in Example 4-3, and PG-210-6 prepared in Example 6-4 were sequentially diluted to various concentrations (PG-210-6 and PG-410 were 0-1,000 nM, and PG-110 and PG-088m were 0-1,507 nM), and then 50 μl of each protein was distributed to the 96-well plate in which the macrophage Raw264.7 was distributed. After 20 minutes, lipopolysaccharide (LPS) was treated in 50 μl increments to a final concentration of 400 ng / ml, and the resultant was cultured at 36°C for 16 hours. The next day, the supernatant was collected and then analyzed by TNF-α ELISA (Biolegend, USA) according to the manufacturer's protocol.

[0237] As a result, Figures 35a-35c As shown, it was found that all fusion proteins containing IL-10Vm according to an exemplary embodiment of the present invention inhibited TNF-α secretion in macrophages in a concentration-dependent manner. No significant difference was found according to the type of fusion protein. The results show that the monomeric IL-10 variant protein (IL-10Vm) according to an exemplary embodiment of the present invention works properly even when the monomeric IL-10 variant protein is linked to the C-terminus of the NTIG protein.

[0238] 5-3: Analysis of IgE binding capacity

[0239] The present inventors analyzed the affinity of the fusion protein (FcεRIα-NTIG-IL-10Vm) prepared in Example 3-3 to mouse IgE and human IgE by biolayer interferometry (BLI).

[0240] For analysis, the inventors first used Dip and Read TMAmine reactive (AR2G) kit (forteBio, catalog number 18-5092) attaches the FcεRIα-NTIG protein without IL-10Vm as a control group and the fusion protein prepared in Example 3-3 to a 96-well plate. Specifically, 200 μl of DW was dispensed into a 96-well plate and then inserted into the amine biosensor contained in the kit for 10 minutes of hydration. Subsequently, after dispensing another 200 μl of DW, EDC and NHS were mixed 1:1 in an amount of 1 / 20 of the required sample amount, and then diluted with DW, and 200 μl of the resultant was dispensed into a 96-well plate. Next, the FcεRIα-modified IgG4 Fc protein and the FcεRIα-NTIG-IL-10Vm fusion protein were diluted to 10 μg / ml in a 10 mM acetate solution at pH 5.0, and then 200 μl of the resultant was added to a 96-well plate. After adding 200 μl of 1 M ethanolamine to the 96-well plate, the biosensor plate and sample plate were placed The instrument was placed in the instrument and the measurement was performed. After the measurement was completed, 200 μl of 1× kinetic buffer was added to the sample plate, and then the baseline was determined. After that, anti-DNP mouse IgE (Sigma) was diluted to various concentrations (50 pM-3.125 nM) with 1× kinetic buffer, 200 μl of the resultant was dispensed to the sample plate, and then the The instrument measures Biolayer Interferometry (BLI).

[0241] [Table 13]

[0242]

[0243] As a result, Fig.36a As shown in Table 13, the K of the fusion protein (FcεRIα-NTIG-IL-10Vm) according to an exemplary embodiment of the present invention is D The value was 0.284 nM, which was slightly lower than that of FcεRIα-modified IgG4 F as the control group. However, the difference was not significant, and thus it was found that the addition of monomeric IL-10 protein did not reduce the binding ability to IgE.

[0244] In addition to the above results, in order to measure the degree of binding between human FcεRIα and human IgE used in the present invention, the present inventors analyzed the affinity of the fusion protein (PG-110) of Example 3-3 and human IgE using the above BLI method.

[0245] [Table 14]

[0246]

[0247]

[0248] As a result, Fig.36b As shown in Table 14, the affinity of the fusion protein according to an exemplary embodiment of the present invention to human IgE is also similar to that to mouse IgE.

[0249] 5-4: Analysis of inhibitory activity against IgE

[0250] Subsequently, the present inventors studied whether the fusion protein (FcεRIα-NTIG-IL-10Vm) prepared in Example 3-3 exhibited inhibitory activity against IgE through in vitro experiments.

[0251] For the purpose of the study, specifically, FcεRIα-modified IgG4 Fc as a control group and the fusion protein (PG-110) of Example 3-3 as an experimental material were sequentially diluted to various concentrations to prepare samples, and the samples were mixed with 1 μl / ml anti-DNP mouse IgE and reacted at room temperature for 30 minutes. Then, bone marrow-derived mast cells obtained from mice were washed with HBSS buffer and adjusted to a concentration of 5×10 5 Cells / 60 μl, and then distributed in 96 well plates. Make 20 μl samples react with IgE and add the test drug to the mast cells prepared previously, and the resultant is cultured in an incubator at 37°C and 5% CO2 for 25 minutes. Next, add 20 μl DNP BSA (500 ng / ml), the resultant is cultured again in an incubator at 37°C and 5% CO2 for 30 minutes, and centrifuge at 4°C at a rate of 1,500 rpm, and separate 30 μl supernatant. Make 30 μl of the separated supernatant and 30 μl substrate (4-nitrophenyl N-acetyl-β-aminoglucosidase, 5.84 mM) mix and culture in an incubator at 37°C and 5% CO2 for 25 minutes. Mix 400 μl 0.1M sodium carbonate buffer (pH is 10) and terminate the reaction. After that, the relative amount of β-hexosaminidase secreted from mast cells was compared by measuring the absorbance at 405 nm, and the mast cell inhibitory effect was examined according to the concentration of each drug. The analysis was performed independently 3 times and the average value was taken.

[0252] As a result, Fig.37 As shown in Table 15, the fusion protein PG-110 according to an exemplary embodiment of the present invention inhibited the activity of IgE in a concentration-dependent manner, and its degree was similar to that of the FcεRIα-modified IgG4Fc as the control group, but the IC 50 Slightly lower than FcεRIα-modified IgG4 Fc.

[0253] [Table 15]

[0254]

[0255] Experimental Example 6: Analysis of binding to CD40L

[0256] The present inventors studied the binding affinity of PG-400 and PG-410 prepared in Examples 4-2 and 4-3 of the present invention, respectively, to human CD40L by BLI analysis.

[0257] For the study, specifically, 200 μl of distilled water was dispensed into a 96-well black flat-bottom plate, the biosensor tray was placed on the hydrated black plate, and then the amine biosensor was inserted for 10 minutes of hydration. Then, 200 μl of distilled water was dispensed into a new 96-well black sample plate, EDC and NHS were mixed 1:1 in an amount of 1 / 20 of the required amount, and then diluted with distilled water, and 200 μl of the resultant was dispensed into the 96-well black sample plate. Next, human CD40L (Peptrotech, Korea) was diluted to 5 μg / ml in a 10 mM acetate solution (pH 6.0), and then 200 μl of the resultant was added to the 96-well black sample plate. 200 μl of 1 M ethanolamine was added to the plate, and the biosensor plate and the sample plate were placed The 96-well black sample plate was placed in the instrument and the binding degree of CD40L was measured based on the amine biosensor. After the measurement was completed, the 96-well black sample plate was taken out from the measuring instrument, 200 μl of 1× kinetic buffer was mixed, and a baseline was set. PG-410 and PG-400 as analytes were sequentially diluted with 1× kinetic buffer so that the concentration was 200-12.5 nM, and then 200 μl of the resultant was dispensed into the 96-well black sample plate. The sample plate was placed again in The binding to CD40L was then measured by BLI analysis.

[0258] [Table 16]

[0259] Analytes PG-400 PG-410 <![CDATA[K D (nM)]]> 0.36nM <1E-12 <![CDATA[K on (1 / Ms)]]> 8.47E+05 1.96E+05 <![CDATA[K dis (1 / s)]]> 3.11E-04 <1E-07

[0260] As a result, Fig.38 As shown in Table 16, the fusion protein (PG-410) according to an exemplary embodiment of the present invention has a K for CD40L on The value is 1 / 4.3 of that of PG-400, but it has a K of fM or less DThe values ​​(<1E-12) indicated very high binding affinity, and thus confirmed that PG-400 and PG-410 proteins can bind well to human CD40L. Here, PG-410, which is a fusion protein (anti-CD40L-NTIG-IL-10Vm) according to an exemplary embodiment of the present invention, was found to bind to CD40L with very high binding affinity.

[0261] Experimental Example 7: Pharmacokinetic Analysis of PG-110

[0262] The present inventors compared the half-life, area under the curve (AUC), and maximum concentration in blood (C max ) etc. to examine the pharmacokinetic characteristics (PK).

[0263] Specifically, fusion protein to be tested (PG-110) is given to 3 male Sprague Dawley (SD) rats in each group with a dosage of 1 mg / kg by subcutaneous (SC), intravenous (IV), intraperitoneal (IP) and intramuscular (IM) routes. Blood was collected at 0.5, 1, 5, 10, 24, 48, 72, 120, 168, 240 and 336 hours before and after injection, and then stored at room temperature for 30 minutes to cause aggregation. Aggregated blood was centrifuged at 3,000 rpm for 10 minutes, and then the serum of each sample was obtained and stored in a cryogenic refrigerator. The concentration of the fusion protein according to an exemplary embodiment of the present invention in serum was analyzed using the IgG-FcQuantitation group (Bethyl, catalog number E80-104, lot number E80-104-30) that can specifically detect the Fc in the protein given.

[0264] As a result, Fig.39 As shown, it was found that the physiologically active protein fused with NTIG can be maintained in the body for a long time regardless of the administration route.

[0265] The present invention has been described with reference to the above-mentioned embodiments and experimental examples, but the description is only exemplary, and those skilled in the art will appreciate that various modifications and other equivalent embodiments may be made therefrom. Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.

[0266] Industry availability

[0267] The NTIG protein according to an exemplary embodiment of the present invention is a fusion partner of various biologically active proteins, and improves the therapeutic effect of biologically active proteins by effectively increasing the half-life of biologically active proteins, and enables safer drug delivery by minimizing effector functions (such as ADCC and CDC, which are shortcomings of Fc fusion proteins). Therefore, the NTIG protein according to an exemplary embodiment of the present invention can be effectively used in the preparation of protein drugs. <110> Progen Ltd. <120> Novel modified immunoglobulin FC fusion protein and its use <130> PT20-5198 <150> US 62 / 847470 <151> 2019-05-14 <160> 77 <170> KoPatentIn 3.0 <210> 1 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Modified IgG4 Fc C-terminal lysine deletion <400> 1 Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys 1 5 10 15 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 20 25 30 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 35 40 45 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 50 55 60 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 65 70 75 80 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 85 90 95 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 100 105 110 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 115 120 125 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 130 135 140 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 145 150 155 160 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 165 170 175 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 180 185 190 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 195 200 205 Leu Ser Leu Ser Leu Gly 210 <210> 2 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Modified IgG4 Fc <400> 2 Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys 1 5 10 15 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 20 25 30 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 35 40 45 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 50 55 60 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 65 70 75 80 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 85 90 95 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 100 105 110 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 115 120 125 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 130 135 140 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 145 150 155 160 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 165 170 175 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 180 185 190 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 195 200 205 Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 3 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Modified IgG4 Fc knob <400> 3 Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys 1 5 10 15 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 20 25 30 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 35 40 45 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 50 55 60 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 65 70 75 80 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 85 90 95 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 100 105 110 Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys 115 120 125 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 130 135 140 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 145 150 155 160 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 165 170 175 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 180 185 190 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 195 200 205 Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 4 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Modified IgG4 Fc pore <400> 4 Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys 1 5 10 15 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 20 25 30 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 35 40 45 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 50 55 60 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 65 70 75 80 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 85 90 95 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 100 105 110 Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 115 120 125 Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp 130 135 140 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 145 150 155 160 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser 165 170 175 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 180 185 190 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 195 200 205 Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 5 <211> 214 <212> PRT <213> Artificial sequence <220> <223> NTIG consensus sequence <220> <221> Variants <222> (18) <223> Asparagine, lysine or glutamine <220> <221> Variants <222> (196) <223> Alanine, phenylalanine, isoleucine, leucine, valine, proline, or tryptophan <400> 5 Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys 1 5 10 15 Asp Xaa Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 20 25 30 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 35 40 45 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 50 55 60 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 65 70 75 80 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 85 90 95 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 100 105 110 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 115 120 125 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 130 135 140 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 145 150 155 160 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 165 170 175 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 180 185 190 Cys Ser Val Xaa His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 195 200 205 Leu Ser Leu Ser Leu Gly 210 <210> 6 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> NTIG C - terminal lysine deletion <400> 6 Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys 1 5 10 15 Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 20 25 30 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 35 40 45 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 50 55 60 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 65 70 75 80 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 85 90 95 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 100 105 110 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 115 120 125 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 130 135 140 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 145 150 155 160 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 165 170 175 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 180 185 190 Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 195 200 205 Leu Ser Leu Ser Leu Gly 210 <210> 7 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> NTIG <400> 7 Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys 1 5 10 15 Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 20 25 30 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 35 40 45 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 50 55 60 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 65 70 75 80 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 85 90 95 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 100 105 110 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 115 120 125 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 130 135 140 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 145 150 155 160 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 165 170 175 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 180 185 190 Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 195 200 205 Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 8 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> NTIG Button <400> 8 Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys 1 5 10 15 Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 20 25 30 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 35 40 45 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 50 55 60 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 65 70 75 80 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 85 90 95 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 100 105 110 Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys 115 120 125 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 130 135 140 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 145 150 155 160 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 165 170 175 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 180 185 190 Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 195 200 205 Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 9 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> NTIG pore <400> 9 Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys 1 5 10 15 Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 20 25 30 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 35 40 45 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 50 55 60 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 65 70 75 80 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 85 90 95 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 100 105 110 Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 115 120 125 Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp 130 135 140 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 145 150 155 160 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser 165 170 175 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 180 185 190 Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 195 200 205 Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 10 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding NTIG with C-terminal lysine deletion <400> 10 tctcataccc agcctctggg cgtgttcctg tttcctccaa agcctaagga ccagctgatg 60 atctctcgga cccctgaagt gacctgcgtg gtggtggatg tgtctcaaga ggaccccgag 120 gtgcagttca attggtacgt ggacggcgtg gaagtgcaca acgccaagac caagcctaga 180 gaggaacagt tcaactccac ctacagagtg gtgtccgtgc tgaccgtgct gcaccaggat 240 tggctgaacg gcaaagagta caagtgcaag gtgtccaaca agggcctgcc ttccagcatc 300 gaaaagacca tctccaaggc taagggccag cctcgggaac ctcaggttta caccctgcct 360 ccaagccaag aggaaatgac caagaaccag gtgtccctga cctgcctggt caagggcttc 420 tacccttccg atatcgccgt ggaatgggag tccaatggcc agcctgagaa caactacaag 480 acaacccctc ctgtgctgga ctccgacggc tccttttttc tgtactcccg cctgaccgtg 540 gacaagtcca gatggcaaga gggcaacgtg ttctcctgct ctgtgctgca cgaggccctg 600 cacaatcact acacccagaa gtccctgtct ctgtctctgg ga 642 <210> 11 <211> 645 <212> DNA <213> Artificial sequence <220> <223> Polynucleotide encoding NTIG <400> 11 tctcataccc agcctctggg cgtgttcctg tttcctccaa agcctaagga ccagctgatg 60 atctctcgga cccctgaagt gacctgcgtg gtggtggatg tgtctcaaga ggaccccgag 120 gtgcagttca attggtacgt ggacggcgtg gaagtgcaca acgccaagac caagcctaga 180 gaggaacagt tcaactccac ctacagagtg gtgtccgtgc tgaccgtgct gcaccaggat 240 tggctgaacg gcaaagagta caagtgcaag gtgtccaaca agggcctgcc ttccagcatc 300 gaaaagacca tctccaaggc taagggccag cctcgggaac ctcaggttta caccctgcct 360 ccaagccaag aggaaatgac caagaaccag gtgtccctga cctgcctggt caagggcttc 420 tacccttccg atatcgccgt ggaatgggag tccaatggcc agcctgagaa caactacaag 480 acaacccctc ctgtgctgga ctccgacggc tccttttttc tgtactcccg cctgaccgtg 540 gacaagtcca gatggcaaga gggcaacgtg ttctcctgct ctgtgctgca cgaggccctg 600 cacaatcact acacccagaa gtccctgtct ctgtctctgg gaaag 645 <210> 12 <211> 645 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding NTIG button <400> 12 tctcacacac agcctctggg cgtgttcctg tttcctccaa agcctaagga ccagctgatg 60 atctctcgga cccctgaagt gacctgcgtg gtggtggatg tgtctcaaga ggaccccgag 120 gtgcagttca attggtacgt ggacggcgtg gaagtgcaca acgccaagac caagcctaga 180 gaggaacagt tcaactccac ctacagagtg gtgtccgtgc tgaccgtgct gcaccaggat 240 tggctgaacg gcaaagagta caagtgcaag gtgtccaaca agggcctgcc tagctccatc 300 gaaaagacca tctccaaggc taagggccag cctcgggaac ctcaggttta caccctgcct 360 ccatgccaag aggaaatgac caagaaccag gtgtccctgt ggtgcctggt caagggcttc 420 tacccttccg acattgccgt ggaatgggag tccaatggcc agcctgagaa caactacaag 480 accacacctc ctgtgctgga ctccgacggc tccttctttc tgtactctcg cctgaccgtg 540 gacaagtcta ggtggcaaga gggcaacgtg ttctcctgct ctgtgctgca cgaggccctg 600 cacaatcact acacccagaa gtccctgtct ctgtccctgg ggaag 645 <210> 13 <211> 645 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding NTIG pore <400> 13 tctcacacac agcccctggg cgtgttcctg tttcctccaa agcctaagga ccagctgatg 60 atctctcgga cccctgaagt gacctgcgtg gtggtggatg tgtctcaaga ggaccccgag 120 gtgcagttca attggtacgt ggacggcgtg gaagtgcaca acgccaagac caagcctaga 180 gaggaacagt tcaactccac ctacagagtg gtgtccgtgc tgaccgtgct gcaccaggat 240 tggctgaacg gcaaagagta caagtgcaag gtgtccaaca agggcctgcc ttccagcatc 300 gaaaagacca tctccaaggc taagggccag cctcgggaac ctcaagtctg taccctgcct 360 cctagccaag aagagatgac caagaaccag gtgtccctgt cttgcgccgt gaagggcttc 420 tacccttccg acattgccgt ggaatgggag tccaatggcc agcctgagaa caactacaag 480 accacacctc ctgtgctgga ctccgacggc tctttctttc tggtgtctcg cctgaccgtg 540 gacaagtcca gatggcaaga gggcaacgtg ttctcctgct ctgtgctgca cgaggccctg 600 cacaatcact acacccagaa gtccctgagc ctgtctctgg gcaag 645 <210> 14 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Linker Peptide <400> 14 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 1 5 10 15 Lys Glu Lys Glu Glu Gln Glu Glu Arg Thr His Thr Cys Pro Pro Cys 20 25 30 Pro <210> 15 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Linker Peptide <400> 15 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Gly Ser Lys Glu Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro 20 25 30 <210> 16 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 16 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 1 5 10 15 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 20 25 30 <210> 17 <211> 26 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 17 Gly Ser Gly Gly Gly Ser Gly Thr Leu Val Thr Val Ser Ser Glu Ser 1 5 10 15 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 20 25 <210> 18 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 18 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 1 5 10 15 Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro 20 25 30 <210> 19 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 19 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 20 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 20 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> twenty one <211> 55 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> twenty one Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala 1 5 10 15 Lys Asn Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Thr Arg Gly Gly 20 25 30 Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg Thr 35 40 45 His Thr Cys Pro Pro Cys Pro 50 55 <210> twenty two <211> 46 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> twenty two Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 15 Glu Ala Ala Ala Lys Ala Leu Glu Ala Glu Ala Ala Ala Lys Glu Ala 20 25 30 Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala 35 40 45 <210> twenty three <211> 5 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> twenty three Gly Gly Gly Gly Ser 1 5 <210> twenty four <211> 6 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> twenty four Gly Ser Ser Gly Gly Ser 1 5 <210> 25 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 25 Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Glu Glu Gln Glu Glu Gly Gly Ser 20 25 <210> 26 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 26 Ala Ala Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 27 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 27 Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10 15 Leu Asp <210> 28 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 28 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 1 5 10 <210> 29 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 29 Gly Ser Ala Gly Ser Ala Ala Gly Ser Gly Glu Phe 1 5 10 <210> 30 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 30 Glu Ala Ala Ala Lys 1 5 <210> 31 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 31 Cys Arg Arg Arg Arg Arg Arg Glu Ala Glu Ala Cys 1 5 10 <210> 32 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 32 Gly Gly Gly Gly Gly Gly Gly Gly 1 5 <210> 33 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 33 Gly Gly Gly Gly Gly Gly 1 5 <210> 34 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 34 Pro Ala Pro Ala Pro 1 5 <210> 35 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 35 Val Ser Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro Asp 1 5 10 15 Val <210> 36 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 36 Pro Leu Gly Leu Trp Ala 1 5 <210> 37 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 37 Thr Arg His Arg Gln Pro Arg Gly Trp Glu 1 5 10 <210> 38 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 38 Ala Gly Asn Arg Val Arg Arg Ser Val Gly 1 5 10 <210> 39 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 39 Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <210> 40 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 40 Gly Ser Ser Gly Gly Ser Gly Ser Ser Gly Gly Ser Gly Gly Gly Asp 1 5 10 15 Glu Ala Asp Gly Ser Arg Gly Ser Gln Lys Ala Gly Val Asp Glu 20 25 30 <210> 41 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 41 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 42 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 42 Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Ala 1 5 10 <210> 43 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 43 Gly Phe Leu Gly 1 <210> 44 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 44 Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly 1 5 10 15 Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg 20 25 30 Glu Thr Lys Thr Pro Glu Cys Pro 35 40 <210> 45 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 45 Gly Gly Ser Gly Gly 1 5 <210> 46 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 46 Gly Gly Ser Gly Gly Ser Gly Gly Ser 1 5 <210> 47 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 47 Gly Gly Gly Ser Gly Gly 1 5 <210> 48 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Linker peptide <400> 48 Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 1 5 10 15 <210> 49 <211> 39 <212> PRT <213> Artificial sequence <220> <223> GLP-1 / exendin hybrid peptide <400> 49 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 50 <211> 287 <212> PRT <213> Artificial Sequence <220> <223> GLP-1E-NTIG <400> 50 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 35 40 45 Ser Gly Gly Gly Gly Ser Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg 50 55 60 Thr His Thr Cys Pro Pro Cys Pro Ser His Thr Gln Pro Leu Gly Val 65 70 75 80 Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr 85 90 95 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 100 105 110 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 115 120 125 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 130 135 140 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 145 150 155 160 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 165 170 175 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 180 185 190 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 195 200 205 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 210 215 220 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 225 230 235 240 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 245 250 255 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu 260 265 270 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 275 280 285 <210> 51 <211> 25 <212> PRT <213> Artificial sequence <220> <223> tPA signal peptide <400> 51 Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser His Ala 20 25 <210> 52 <211> 33 <212> PRT <213> Artificial sequence <220> <223> GLP-2-2G (teduglutide) <400> 52 His Gly Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp <210> 53 <211> 281 <212> PRT <213> Artificial sequence <220> <223> GLP-2-2G-NTIG <400> 53 His Gly Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 35 40 45 Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg Thr His Thr Cys Pro Pro 50 55 60 Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys 65 70 75 80 Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 85 90 95 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 100 105 110 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 115 120 125 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 130 135 140 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 145 150 155 160 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 165 170 175 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 180 185 190 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 195 200 205 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 210 215 220 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 225 230 235 240 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 245 250 255 Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln 260 265 270 Lys Ser Leu Ser Leu Ser Leu Gly Lys 275 280 <210> 54 <211> 160 <212> PRT <213> Artificial Sequence <220> <223> IL-10V <400> 54 Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro 1 5 10 15 Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg 20 25 30 Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu 35 40 45 Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala 50 55 60 Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala 65 70 75 80 Glu Asn Gln Asp Pro Asp Ala Lys Ala His Val Asn Ser Leu Gly Glu 85 90 95 Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu 100 105 110 Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe 115 120 125 Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp 130 135 140 Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn 145 150 155 160 <210> 55 <211> 425 <212> PRT <213> Artificial sequence <220> <223> NTIG-IL-10V <400> 55 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 1 5 10 15 Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ser His 20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys Ala Ala Gly Ser Gly Gly Gly Gly Gly Ser Gly 245 250 255 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Pro Gly Gln Gly Thr Gln 260 265 270 Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu 275 280 285 Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met 290 295 300 Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp 305 310 315 320 Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe 325 330 335 Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ala 340 345 350 Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu 355 360 365 Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys 370 375 380 Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly 385 390 395 400 Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu 405 410 415 Ala Tyr Met Thr Met Lys Ile Arg Asn 420 425 <210> 56 <211> 169 <212> PRT <213> Artificial Sequence <220> <223> IL-10Vm <400> 56 Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro 1 5 10 15 Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg 20 25 30 Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu 35 40 45 Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala 50 55 60 Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala 65 70 75 80 Glu Asn Gln Asp Pro Asp Ala Lys Ala His Val Asn Ser Leu Gly Glu 85 90 95 Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu 100 105 110 Pro Cys Glu Asn Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys Ser Lys 115 120 125 Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly 130 135 140 Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu 145 150 155 160 Ala Tyr Met Thr Met Lys Ile Arg Asn 165 <210> 57 <211> 403 <212> PRT <213> Artificial sequence <220> <223> NTIG-IL-10Vm <400> 57 Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys 1 5 10 15 Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 20 25 30 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 35 40 45 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 50 55 60 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 65 70 75 80 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 85 90 95 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 100 105 110 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 115 120 125 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 130 135 140 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 145 150 155 160 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 165 170 175 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 180 185 190 Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 195 200 205 Leu Ser Leu Ser Leu Gly Ala Ala Gly Ser Gly Gly Gly Gly Gly Ser 210 215 220 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Pro Gly Gln Gly Thr 225 230 235 240 Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met 245 250 255 Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln 260 265 270 Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu 275 280 285 Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln 290 295 300 Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp 305 310 315 320 Ala Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg 325 330 335 Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Gly Gly 340 345 350 Ser Gly Gly Ser Gly Gly Ser Lys Ser Lys Ala Val Glu Gln Val Lys 355 360 365 Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser 370 375 380 Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys 385 390 395 400 Ile Arg Asn <210> 58 <211> 180 <212> PRT <213> Artificial Sequence <220> <223> FceRIa <400> 58 Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile 1 5 10 15 Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe 20 25 30 Glu Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu 35 40 45 Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly 50 55 60 Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr 65 70 75 80 Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu Val 85 90 95 Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp Arg Asn 100 105 110 Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala Leu Lys 115 120 125 Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr Val Glu 130 135 140 Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr 145 150 155 160 Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Arg Glu Lys 165 170 175 Tyr Trp Leu Gln 180 <210> 59 <211> 605 <212> PRT <213> Artificial Sequence <220> <223> FceRIa-NTIG-IL-10V <400> 59 Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile 1 5 10 15 Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe 20 25 30 Glu Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu 35 40 45 Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly 50 55 60 Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr 65 70 75 80 Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu Val 85 90 95 Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp Arg Asn 100 105 110 Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala Leu Lys 115 120 125 Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr Val Glu 130 135 140 Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr 145 150 155 160 Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Arg Glu Lys 165 170 175 Tyr Trp Leu Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 180 185 190 Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro 195 200 205 Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys 210 215 220 Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 225 230 235 240 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 245 250 255 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 260 265 270 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 275 280 285 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 290 295 300 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 305 310 315 320 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 325 330 335 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 340 345 350 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 355 360 365 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 370 375 380 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 385 390 395 400 Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln 405 410 415 Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ala Gly Ser Gly Gly Gly 420 425 430 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Pro Gly 435 440 445 Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu 450 455 460 Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr 465 470 475 480 Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser 485 490 495 Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu 500 505 510 Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln 515 520 525 Asp Pro Asp Ala Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys 530 535 540 Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu 545 550 555 560 Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu 565 570 575 Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile 580 585 590 Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn 595 600 605 <210> 60 <211> 613 <212> PRT <213> Artificial Sequence <220> <223> FceRIa-NTIG-IL-10Vm <400> 60 Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile 1 5 10 15 Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe 20 25 30 Glu Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu 35 40 45 Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly 50 55 60 Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr 65 70 75 80 Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu Val 85 90 95 Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp Arg Asn 100 105 110 Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala Leu Lys 115 120 125 Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr Val Glu 130 135 140 Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr 145 150 155 160 Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Arg Glu Lys 165 170 175 Tyr Trp Leu Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 180 185 190 Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro 195 200 205 Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys 210 215 220 Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 225 230 235 240 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 245 250 255 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 260 265 270 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 275 280 285 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 290 295 300 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 305 310 315 320 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 325 330 335 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 340 345 350 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 355 360 365 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 370 375 380 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 385 390 395 400 Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln 405 410 415 Lys Ser Leu Ser Leu Ser Leu Gly Ala Ala Gly Ser Gly Gly Gly Gly 420 425 430 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Pro Gly Gln 435 440 445 Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro 450 455 460 Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe 465 470 475 480 Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu 485 490 495 Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met 500 505 510 Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp 515 520 525 Pro Asp Ala Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr 530 535 540 Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn 545 550 555 560 Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys Ser Lys Ala Val Glu Gln 565 570 575 Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala 580 585 590 Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr 595 600 605 Met Lys Ile Arg Asn 610 <210> 61 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Chimeric anti - CD40L heavy chain VH - CH1 <400> 61 Glu Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Tyr Met Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ile Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Asp Gly Arg Asn Asp Met Asp Ser Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val 210 215 <210> 62 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> Chimeric Anti-CD40L Light Chain <400> 62 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Arg Val Ser Ser Ser 20 25 30 Thr Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ser Ala Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95 Glu Ile Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Ala Asp Ala Ala Pro Thr Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 63 <211> 19 <212> PRT <213> Artificial sequence <220> <223> IgG1 heavy chain signal sequence <400> 63 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser <210> 64 <211> 216 <212> PRT <213> Artificial sequence <220> <223> Humanized anti-CD40L heavy chain VH-CH1 <400> 64 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Tyr Met Tyr Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Asp Gly Arg Asn Asp Met Asp Ser Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val 210 215 <210> 65 <211> 230 <212> PRT <213> Artificial Sequence <220> <223> Hinge + NTIG <400> 65 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ser 1 5 10 15 His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 20 25 30 Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 35 40 45 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 50 55 60 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 65 70 75 80 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 85 90 95 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 100 105 110 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 115 120 125 Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn 130 135 140 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 145 150 155 160 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 165 170 175 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 180 185 190 Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 195 200 205 Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 210 215 220 Ser Leu Ser Leu Gly Lys 225 230 <210> 66 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> IgG1 Light Chain Signal Sequence <400> 66 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly 20 <210> 67 <211> 218 <212> PRT <213> Artificial sequence <220> <223> Humanized anti-CD40L light chain <400> 67 Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Arg Val Ser Ser Ser 20 25 30 Thr Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Val Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95 Glu Ile Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 68 <211> 229 <212> PRT <213> Artificial sequence <220> <223> Hinge + NTIG lysine deletion <400> 68 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ser 1 5 10 15 His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 20 25 30 Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 35 40 45 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 50 55 60 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 65 70 75 80 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 85 90 95 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 100 105 110 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 115 120 125 Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn 130 135 140 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 145 150 155 160 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 165 170 175 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 180 185 190 Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 195 200 205 Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 210 215 220 Ser Leu Ser Leu Gly 225 <210> 69 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Humanized anti - CD40L VL <400> 69 Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Arg Val Ser Ser Ser 20 25 30 Thr Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Val Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95 Glu Ile Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val 115 <210> 70 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Humanized anti-CD40L VH <400> 70 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Tyr Met Tyr Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Asp Gly Arg Asn Asp Met Asp Ser Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 71 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> GLP-1E-NTIG Button Glycan Linker <400> 71 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser Gly Gly Gly Gly Ser Ala Lys Asn Thr Thr Ala Pro Ala Thr 50 55 60 Thr Arg Asn Thr Thr Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu 65 70 75 80 Lys Glu Glu Gln Glu Glu Arg Thr His Thr Cys Pro Pro Cys Pro Ser 85 90 95 His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 100 105 110 Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 115 120 125 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 130 135 140 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 145 150 155 160 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 165 170 175 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 180 185 190 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 195 200 205 Pro Gln Val Tyr Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys Asn 210 215 220 Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 225 230 235 240 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 245 250 255 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 260 265 270 Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 275 280 285 Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 290 295 300 Ser Leu Ser Leu Gly Lys 305 310 <210> 72 <211> 281 <212> PRT <213> Artificial sequence <220> <223> GLP-2-2G-NTIG pore non-glycan linker <400> 72 His Gly Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 35 40 45 Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg Thr His Thr Cys Pro Pro 50 55 60 Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys 65 70 75 80 Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 85 90 95 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 100 105 110 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 115 120 125 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 130 135 140 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 145 150 155 160 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 165 170 175 Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met 180 185 190 Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro 195 200 205 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 210 215 220 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 225 230 235 240 Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 245 250 255 Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln 260 265 270 Lys Ser Leu Ser Leu Ser Leu Gly Lys 275 280 <210> 73 <211> 287 <212> PRT <213> Artificial sequence <220> <223> GLP-1E-NTIG nonasaccharide linker <400> 73 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 35 40 45 Ser Gly Gly Gly Gly Ser Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg 50 55 60 Thr His Thr Cys Pro Pro Cys Pro Ser His Thr Gln Pro Leu Gly Val 65 70 75 80 Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr 85 90 95 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 100 105 110 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 115 120 125 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 130 135 140 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 145 150 155 160 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 165 170 175 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 180 185 190 Pro Cys Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 195 200 205 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 210 215 220 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 225 230 235 240 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 245 250 255 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu 260 265 270 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 275 280 285 <210> 74 <211> 314 <212> PRT <213> Artificial sequence <220> <223> IL-13Rα2 <400> 74 Asp Thr Glu Ile Lys Val Asn Pro Pro Gln Asp Phe Glu Ile Val Asp 1 5 10 15 Pro Gly Tyr Leu Gly Tyr Leu Tyr Leu Gln Trp Gln Pro Pro Leu Ser 20 25 30 Leu Asp His Phe Lys Glu Cys Thr Val Glu Tyr Glu Leu Lys Tyr Arg 35 40 45 Asn Ile Gly Ser Glu Thr Trp Lys Thr Ile Ile Thr Lys Asn Leu His 50 55 60 Tyr Lys Asp Gly Phe Asp Leu Asn Lys Gly Ile Glu Ala Lys Ile His 65 70 75 80 Thr Leu Leu Pro Trp Gln Cys Thr Asn Gly Ser Glu Val Gln Ser Ser 85 90 95 Trp Ala Glu Thr Thr Tyr Trp Ile Ser Pro Gln Gly Ile Pro Glu Thr 100 105 110 Lys Val Gln Asp Met Asp Cys Val Tyr Tyr Asn Trp Gln Tyr Leu Leu 115 120 125 Cys Ser Trp Lys Pro Gly Ile Gly Val Leu Leu Asp Thr Asn Tyr Asn 130 135 140 Leu Phe Tyr Trp Tyr Glu Gly Leu Asp His Ala Leu Gln Cys Val Asp 145 150 155 160 Tyr Ile Lys Ala Asp Gly Gln Asn Ile Gly Cys Arg Phe Pro Tyr Leu 165 170 175 Glu Ala Ser Asp Tyr Lys Asp Phe Tyr Ile Cys Val Asn Gly Ser Ser 180 185 190 Glu Asn Lys Pro Ile Arg Ser Ser Tyr Phe Thr Phe Gln Leu Gln Asn 195 200 205 Ile Val Lys Pro Leu Pro Pro Val Tyr Leu Thr Phe Thr Arg Glu Ser 210 215 220 Ser Cys Glu Ile Lys Leu Lys Trp Ser Ile Pro Leu Gly Pro Ile Pro 225 230 235 240 Ala Arg Cys Phe Asp Tyr Glu Ile Glu Ile Arg Glu Asp Asp Thr Thr 245 250 255 Leu Val Thr Ala Thr Val Glu Asn Glu Thr Tyr Thr Leu Lys Thr Thr 260 265 270 Asn Glu Thr Arg Gln Leu Cys Phe Val Val Arg Ser Lys Val Asn Ile 275 280 285 Tyr Cys Ser Asp Asp Gly Ile Trp Ser Glu Trp Ser Asp Lys Gln Cys 290 295 300 Trp Glu Gly Glu Asp Leu Ser Lys Lys Thr 305 310 <210> 75 <211> 160 <212> PRT <213> Artificial sequence <220> <223> IL-10wt <400> 75 Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro 1 5 10 15 Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg 20 25 30 Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu 35 40 45 Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala 50 55 60 Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala 65 70 75 80 Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu 85 90 95 Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu 100 105 110 Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe 115 120 125 Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp 130 135 140 Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn 145 150 155 160 <210> 76 <211> 33 <212> PRT <213> Artificial sequence <220> <223> GLP-2-2 (3-point mutation) <400> 76 His Gly Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Gly 1 5 10 15 Gln Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp <210> 77 <211> 103 <212> PRT <213> Artificial sequence <220> <223> IgG1 CH3 <400> 77 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 1 5 10 15 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 20 25 30 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 35 40 45 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 50 55 60 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 65 70 75 80 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 85 90 95 Leu Ser Leu Ser Pro Gly Lys 100

Claims

1. An immunoglobulin Fc domain variant protein represented by an amino acid sequence selected from SEQ ID NO: 6-9.

2. A fusion protein, wherein at least one biologically active protein is linked to the N-terminus and / or C-terminus of the immunoglobulin Fc domain variant protein of claim 1, wherein the biologically active protein is Fab, scFv, diabody, triabody, sdAb, V H H, GLP-1 analogs, GLP-2 analogs, IL-10 or Fc receptor.

3. The fusion protein of claim 2, wherein the biologically active protein is linked to the N-terminus or C-terminus of the immunoglobulin Fc domain variant protein through a linker peptide or a hinge region of an antibody.

4. A polynucleotide encoding the immunoglobulin Fc domain variant protein of claim 1 or the fusion protein of claim 2.

5. A recombinant vector comprising the polynucleotide of claim 4.

6. A homodimer comprising the fusion protein of claim 2.

7. A heterodimer comprising the fusion protein of claim 2.

8. The heterodimer of claim 7, wherein the immunoglobulin Fc domain variant protein has a knob-into-hole structure.

9. A composition comprising at least one selected from the group consisting of the fusion protein of claim 2, the homodimer of claim 6 and the heterodimer of claim 7 as an active ingredient.

Images